TWI805542B

TWI805542B - Peptides and peptidomimetics in combination with t cell activating and/or checkpoint inhibiting agents for cancer treatment

Info

Publication number: TWI805542B
Application number: TW105134194A
Authority: TW
Inventors: 河邊拓己
Original assignee: 日商坎巴斯有限公司
Priority date: 2015-10-23
Filing date: 2016-10-21
Publication date: 2023-06-21
Also published as: TW201729827A

Abstract

This invention provides compounds including peptides and peptidomimetics that can be used to treat cell proliferative disorders, such as those associated with benign and malignant tumor cells, and combinations of T cell activating agents and/or an immune checkpoint inhibitors with and without peptides and peptidimimetics. The invention compounds and combinations can be used to inhibit cell growth, such as treat a tumor or cancer.

Description

Combinations of peptides and peptidomimetics with T cell activators and/or checkpoint inhibitors for cancer therapy

本發明係關於單獨及與活化T細胞或為查核點抑制劑之治療組合具有抗細胞增殖活性之化合物(包括肽及擬肽)。因此，本發明化合物可用於抑制細胞增殖及因此治療細胞增殖病症(包括癌症)。 The present invention relates to compounds (including peptides and peptidomimetics) having anti-cell proliferative activity alone and in combination with treatments that activate T cells or are checkpoint inhibitors. Accordingly, the compounds of the invention are useful for inhibiting cell proliferation and thus treating cell proliferative disorders, including cancer.

細胞週期包含S期(DNA複製)、M期(有絲分裂)及在S期與M期之間之兩個間隔期(G1及G2期)。細胞週期中之查核點確保準確進展，例如監測DNA完整性之狀態、DNA複製、細胞大小及周圍環境(Maller,J.L.Curr.Opin.Cell Biol.,3：26(1991))。對於多細胞生物體而言維持基因體之完整性尤為重要，且存在多個監測基因體之狀態之查核點。尤其係分別在DNA複製及有絲分裂之前存在之G1查核點及G2查核點。在進入s期前校正DNA損害係至關重要的，乃因在受損DNA經複製後，其通常引起突變(Hartwell,L.Cell,71：543(1992))。前進穿過G1及G2查核點而不修復廣泛的DNA損害可誘導細胞凋亡及/或劇變。 The cell cycle consists of S phase (DNA replication), M phase (mitosis) and two intervals (G1 and G2 phases) between S and M phases. Checkpoints in the cell cycle ensure accurate progression, such as monitoring the status of DNA integrity, DNA replication, cell size and surrounding environment (Maller, J.L. Curr. Opin. Cell Biol., 3:26 (1991)). Maintaining the integrity of the genome is particularly important for multicellular organisms, and there are multiple checkpoints that monitor the state of the genome. In particular, the G1 checkpoint and the G2 checkpoint exist before DNA replication and mitosis, respectively. Correction of DNA damage before entering s phase is critical because damaged DNA often causes mutations after replication (Hartwell, L. Cell, 71:543 (1992)). Progression through the G1 and G2 checkpoints without repair of extensive DNA damage can induce apoptosis and/or catastrophe.

大多數癌細胞均攜載G1查核點相關性蛋白質(例如p53、Rb、MDM-2、p16^INK4及p19^ARF)之異常(Levine,A.J.Cell,88：323(1997))。或者，突變可導致致癌基因產物(例如Ras、MDM-2及週期蛋白D)過表現及/或過活化，從而降低G1查核點之嚴格度。除該等突變以外，過量生長因子信號傳導可由生長因子之過表現造成且可降低G1查核點之嚴格度。與功能型突變之喪失及獲得一起，生長因子受體或下游信號轉導分子之連續活化可因使G1查核點無效而引起細胞轉化。經廢除G1查核點促成較高突變率及在癌細胞中觀察到之許多突變。因此，大多數癌細胞依賴於G2查核點來抵抗過度DNA損害而存活(O’Connor及Fan，Prog.Cell Cycle Res.,2：165(1996))。 Most cancer cells carry abnormalities in G1 checkpoint-associated proteins such as p53, Rb, MDM-2, p16 ^INK4 , and p19 ^ARF (Levine, AJ Cell, 88:323 (1997)). Alternatively, mutations can lead to overexpression and/or overactivation of oncogene products (eg, Ras, MDM-2, and cyclin D), thereby reducing the stringency of the G1 checkpoint. In addition to such mutations, excess growth factor signaling can result from overexpression of growth factors and can reduce the stringency of the G1 checkpoint. Together with loss and gain-of-function mutations, sequential activation of growth factor receptors or downstream signaling molecules can lead to cellular transformation by inactivating the G1 checkpoint. Abrogation of the G1 checkpoint contributes to the higher mutation rate and many mutations observed in cancer cells. Thus, most cancer cells depend on the G2 checkpoint for survival against excessive DNA damage (O'Connor and Fan, Prog. Cell Cycle Res., 2:165 (1996)).

相信在DNA損害後促進細胞週期G2停滯之機制在自酵母至人類之物種中係保守的。在受損DNA存在下，Cdc2/週期蛋白B激酶由於Cdc2激酶上蘇胺酸-14及酪胺酸-15殘基之抑制性磷酸化而保持無活性或週期蛋白B之蛋白質含量降低。在有絲分裂開始時，雙重磷酸酶Cdc25去除該等抑制性磷酸酯且藉此活化Cdc2/週期蛋白B激酶。Cdc2/週期蛋白B之活化等效於M期開始。 The mechanisms that promote cell cycle G2 arrest following DNA damage are believed to be conserved in species from yeast to humans. In the presence of damaged DNA, the Cdc2/cyclin B kinase remains inactive or the protein content of cyclin B is reduced due to inhibitory phosphorylation of threonine-14 and tyrosine-15 residues on the Cdc2 kinase. At the onset of mitosis, the dual phosphatase Cdc25 removes these inhibitory phosphates and thereby activates the Cdc2/cyclin B kinase. Activation of Cdc2/cyclin B is equivalent to the onset of M phase.

在裂殖酵母中，蛋白激酶Chk1係因應受損DNA之細胞週期停滯所必需的。Chk1激酶在若干rad基因產物之下游起作用且藉由DNA損害後之磷酸化而改質。已知出芽酵母之激酶Rad53及裂殖酵母之Cds1傳導來自未複製DNA之信號。在Chk1與Cds1之間似乎存在一些冗餘性，因為Chk1及Cds1之消除最終導致受損DNA誘導之G2停滯中斷。有趣地，Chk1及Cds1二者均磷酸化Cdc25且促進Rad24結合至Cdc25，從而使Cdc25隔絕至胞質液且防止Cdc2/週期蛋白B活化。因此，Cdc25似乎係該等激酶之共同靶標，暗指此分子係G2查核點中不可或缺的因子。 In fission yeast, the protein kinase Chk1 is required for cell cycle arrest in response to damaged DNA. Chk1 kinase acts downstream of several rad gene products and is modified by phosphorylation following DNA damage. The kinase Rad53 in budding yeast and Cds1 in fission yeast are known to transmit signals from unreplicated DNA. There appears to be some redundancy between Chk1 and Cds1, as elimination of Chk1 and Cds1 ultimately leads to disruption of damaged DNA-induced G2 arrest. Interestingly, both Chk1 and Cds1 phosphorylate Cdc25 and promote the binding of Rad24 to Cdc25, sequestering Cdc25 to the cytosol and preventing Cdc2/cyclin B activation. Thus, Cdc25 appears to be a common target of these kinases, implying that this molecule is an indispensable factor in the G2 checkpoint.

在人類中，hChk1(裂殖酵母Chk1之人類同系物)及Chk2/HuCds1(出芽酵母Rad53及裂殖酵母Cds1之人類同系物)因應DNA損害而在絲胺酸-216(關鍵調節位點)處磷酸化Cdc25C。此磷酸化為小酸性蛋白質14-3-3(裂殖酵母之Rad24及Rad25之人類同系物)產生結合位點。此磷酸化之調節作用可由以下事實明確指示：在Cdc25C上絲胺酸-216取代為丙胺酸中斷人類細胞中之細胞週期G2停滯。然而，並未完全瞭解G2查核點之機制。 In humans, hChk1 (human homologue of fission yeast Chk1) and Chk2/HuCds1 (ex. Budding yeast Rad53 and the human homologue of fission yeast Cds1) phosphorylate Cdc25C at serine-216, a key regulatory site, in response to DNA damage. This phosphorylation creates a binding site for the small acidic protein 14-3-3, the human homologues of Rad24 and Rad25 of fission yeast. Regulation of this phosphorylation is clearly indicated by the fact that substitution of serine-216 to alanine on Cdc25C interrupts cell cycle G2 arrest in human cells. However, the mechanism of the G2 checkpoint is not fully understood.

腫瘤微環境亦在預防或促進癌症細胞生長、侵襲、轉移及抗腫瘤免疫性中起作用，其影響患者預後。曾經預期對抗癌細胞之巨噬細胞已指示在腫瘤發展中起抑制及促進作用。具有古典抗腫瘤表型之巨噬細胞稱作M1，其具有促發炎性，且具有促腫瘤及消炎類型之彼等稱作M2，且在此類別內具有至少三種主要亞型(Martinez及Gordon,F1000Prime Reports 6：13(2014))。 The tumor microenvironment also plays a role in preventing or promoting cancer cell growth, invasion, metastasis and anti-tumor immunity, which affects patient prognosis. Macrophages, once expected to fight cancer cells, have been indicated to play both suppressive and promoting roles in tumor development. Macrophages with a classical anti-tumor phenotype are called M1, which are pro-inflammatory, and those with a pro-tumor and anti-inflammatory type are called M2, and within this class there are at least three major subtypes (Martinez and Gordon, F1000 Prime Reports 6:13 (2014)).

嗜中性球細胞外陷阱(NET)連同白血球一起代表腫瘤微環境之另一要素。儘管NET之形成對於嗜中性球對抗侵襲性微生物係有用的，但該等NET可在癌症患者中促成深層靜脈血栓形成(DVT)(Martinnod及Wanger,Blood(2013))及腫瘤細胞轉移(Cools-Lartigue,J.等人，J.Clin.Invest.(2013))。因此，NET可不利地影響患者存活。DVT在癌症患者中較為常見且潛在地致死，且白血球增多症(其導致高WBC)係主要風險因素(Pabinger,I.等人，Blood 122：12(2013)；Blix,K.等人，PLOS One 4：8(2013)；Wang,T.F.等人，Thromb.Res.133(1)：25(2014))。 Along with leukocytes, neutrophil extracellular traps (NETs) represent another element of the tumor microenvironment. Although NET formation is useful for neutrophils against invasive microorganisms, these NETs can contribute to deep vein thrombosis (DVT) (Martinnod and Wanger, Blood (2013)) and tumor cell metastasis in cancer patients (Cools -Lartigue, J. et al., J. Clin. Invest. (2013)). Thus, NETs can adversely affect patient survival. DVT is common and potentially fatal in cancer patients, and leukocytosis (which results in high WBC) is a major risk factor (Pabinger, I. et al., Blood 122:12 (2013); Blix, K. et al., PLOS One 4:8 (2013); Wang, T.F. et al., Thromb. Res. 133(1):25 (2014)).

本發明提供具有一或多種如下活性之化合物之組合之方法及用途：抑制細胞增殖，刺激細胞凋亡或劇變，或廢除細胞之細胞週期G2查核點；或治療(例如)特徵在於細胞增殖病症之不合意細胞增殖或存活。舉例而言，本發明提供如下方法及用途：抑制細胞增殖；廢除細胞之細胞週期G2查核點；增加細胞對核酸損害劑或治療之敏感性；增加對細胞之核酸損害。 The invention provides methods and uses of combinations of compounds having one or more of the following activities: inhibiting cell proliferation, stimulating cell apoptosis or catastrophe, or abolishing the cell cycle G2 checkpoint of cells; or treating, for example, a cell proliferative disorder characterized by Undesirable cell proliferation or survival. For example, The present invention provides the following methods and uses: inhibiting cell proliferation; abolishing the cell cycle G2 checkpoint of cells; increasing the sensitivity of cells to nucleic acid damaging agents or treatments; increasing nucleic acid damage to cells.

在一個實施例中，在哺乳動物中用於增加過度增殖細胞之核酸損害或用於預防或治療細胞增殖病症之方法或用途包括向哺乳動物投與T細胞活化劑及免疫查核點抑制劑。 In one embodiment, a method or use for increasing nucleic acid damage in hyperproliferative cells or for preventing or treating a cell proliferative disorder in a mammal comprises administering a T cell activator and an immune checkpoint inhibitor to the mammal.

在另一實施例中，用於增加過度增殖細胞之核酸損害或用於預防或治療哺乳動物(例如，人類)之細胞增殖病症之方法或用途，包括向哺乳動物投與T細胞活化劑及/或免疫查核點抑制劑及肽化合物，其中該肽化合物包含以下序列中之任一者：A)包含表示為P1-P6之殘基且具有結構P1、P2、P3、P4、P5、P6或P6、P5、P4、P3、P2、P1之肽；其中P1係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、佔據類似側鏈空間之胺基酸，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉、喹唑啉基團之任一胺基酸；其中P2係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、Bpa、Phe4NO2、佔據類似側鏈空間之胺基酸，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉或喹唑啉基團之任一胺基酸；其中P3、P4、P5係任一胺基酸，或其中P3、P4、P5中之一或多者係簡單碳鏈，使得P2與P6之間之距離與當P3、P4、P5中之每一者係胺基酸時之距離大約相同；其中P6係Bpa、Phe4NO2、任一種胺基酸及Tyr、任一種胺基酸及Phe、任一胺基酸或不存在；B)或A)之肽，其中具有簡單碳鏈之胺基酸係11-胺基十一酸、10-胺基癸酸、9-胺基壬酸、8-胺基辛酸、 7-胺基庚酸、6-胺基己酸或具有一或多個不飽和碳鍵之類似結構，及/或其中該任一種胺基酸係Ser，及/或其中P4係Trp，及/或其中佔據類似側鏈空間之該胺基酸係Tyr或Phe；或包含表示為P1-P12之殘基之肽，其具有任一以下結構：P1、P2、P3、P4、P5、P6；P6、P5、P4、P3、P2、P1；P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12；P1、P2、P3、P4、P5、P6、P12、P11、P10、P9、P8、P7；P6、P5、P4、P3、P2、P1、P7、P8、P9、P10、P11、P12；P6、P5、P4、P3、P2、P1、P12、P11、P10、P9、P8、P7；P7、P8、P9、P10、P11、P12、P1、P2、P3、P4、P5、P6；P7、P8、P9、P10、P11、P12、P6、P5、P4、P3、P2、P1；P12、P11、P10、P9、P8、P7、P1、P2、P3、P4、P5、P6；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1；P12、P11、P6、P9、P8、P7、P2、P1；P12、P11、P10、P6、P9、P4、P7、P2、P1；P1、P2、P7、P8、P9、P6、P11、P12；或P1、P2、P7、P4、P9、P6、P10、P11、P12；其中P1係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、Bpa、Phe4NO2、佔據類似側鏈空間之胺基酸(例如d-Tyr或l-Tyr、d-Phe或l-Phe)，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉或喹唑啉基團之任一胺基酸；其中P2係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)或佔據類似側鏈空間之胺基酸，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

烷、喹喏啉、喹唑啉基團之任一胺基酸；其中P3、P4、 P5係任一胺基酸，或其中P3、P4、P5中之一或多者係簡單碳鏈，使得P2與P6之間之距離與當P3、P4、P5中之每一者係胺基酸時之距離大約相同；其中P6係Bpa、Phe4NO2、任一種胺基酸及Tyr、任一種胺基酸及Phe；且其中P7、P8、P9、P10、P11、P12中之至少三者係鹼性胺基酸，且其餘為任一胺基酸或不存在；或C)之肽，其中具有簡單碳鏈之胺基酸係11-胺基十一酸、10-胺基癸酸、9-胺基壬酸、8-胺基辛酸、7-胺基庚酸、6-胺基己酸或具有一或多個不飽和碳鍵之類似結構，及/或，其中該任一種胺基酸係Ser，及/或，其中P4係Trp，及/或，其中佔據類似側鏈空間之該胺基酸係Tyr或Phe；或包含表示為P1-P12之殘基之肽，其具有任一以下結構：P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1；P12、P11、P10、P6、P9、P4、P7、P2、P1；或P1、P2、P7、P4、P9、P6、P10、P11、P12；其中P1係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、Bpa、Phe4NO2、佔據類似側鏈空間之胺基酸，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉或喹唑啉基團之任一胺基酸；其中P2係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、佔據類似側鏈空間之胺基酸，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉、喹唑啉基團之任一胺基酸；其中P3、P4、P5係任一胺基酸，或其中P3、P4、P5中之一或多者係簡單碳鏈，使得P2與P6之間之距離與當P3、P4、P5中之每一者係胺基酸時之距離大約相同；其中P6係Bpa、 Phe4NO2、任一種胺基酸及Tyr、任一種胺基酸及Phe、任一胺基酸或不存在；且其中P7、P8、P9、P10、P11、P12中之至少三者係鹼性胺基酸，且其餘為任一胺基酸或不存在；或E)之肽，其中具有簡單碳鏈之胺基酸係胺基十一酸或8-胺基辛酸，及/或，其中該任一種胺基酸係Ser，及/或，其中佔據類似側鏈空間之該胺基酸係Tyr或Phe；或包含表示為P1-P12之殘基之肽，其具有任一以下結構：P1、P2、P3、P4、P5、P6或P6、P5、P4、P3、P2、P1，其中P1係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、Bpa、Phe4NO2、Tyr或Phe；其中P2係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、Bpa、Phe4NO2、Tyr或Phe；其中P3係Ser、Arg、Cys、Pro或Asn；其中P4係Trp；其中P5係Ser、Arg或Asn；或其中P3、P4、P5係單一胺基十一酸或單一8-胺基辛酸；且其中P6係Bpa、Phe4NO2、(Ser-Tyr)或(Ser-Phe)；或包含表示為P1-P12之殘基之肽，其具有任一以下結構：P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12；P1、P2、P3、P4、P5、P6、P12、P11、P10、P9、P8、P7；P6、P5、P4、P3、P2、P1、P7、P8、P9、P10、P11、P12；P6、P5、P4、P3、P2、P1、P12、P11、P10、P9、P8、P7；P7、P8、P9、P10、P11、P12、P1、P2、P3、P4、P5、P6；P7、P8、P9、P10、P11、P12、P6、P5、P4、P3、P2、P1；P12、P11、P10、P9、P8、P7、P1、P2、P3、P4、P5、P6；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1；P12、P11、P6、P9、P8、P7、P2、P1；P12、P11、P10、P6、P9、P4、P7、P2、P1；P1、P2、P7、P8、P9、P6、P11、P12；或P1、P2、P7、P4、P9、P6、P10、P11、P12；其中P1係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、 Bpa、Phe4NO2、Tyr或Phe；其中P2係Cha、Nal(2)、(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)、Bpa、Phe4NO2、Tyr或Phe；其中P3係Ser、Arg、Cys、Pro或Asn；其中P4係Trp；其中P5係Ser、Arg或Asn；或其中P3、P4、P5係單一胺基十一酸或單一8-胺基辛酸；其中P6係Bpa、Phe4NO2、(d-Ser-d-Tyr)或(d-Ser-d-Phe)；且其中P7、P8、P9、P10、P11、P12中之至少三者係Arg或Lys，且其餘為任一胺基酸或不存在；或包含表示為P1-P12之殘基之肽，其具有任一以下結構：P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1；P12、P11、P10、P6、P9、P4、P7、P2、P1；或P1、P2、P7、P4、P9、P6、P10、P11、P12；其中P1係Cha或Nal(2)；其中P2係(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)、(Phe-4CF3)；其中P3係Ser；其中P4係Trp；其中P5係Ser或Asn；其中P6係Bpa、Phe4NO2、(Ser-Tyr)或(Ser-Phe)；且其中P7、P8、P9、P10、P11、P12中之至少三者係Arg，且其餘為任一胺基酸或不存在；或包含表示為P1-P12之殘基之肽，其具有任一以下結構：P1、P2、P3、P4、P5、P6或P6、P5、P4、P3、P2、P1；其中P1係Cha或Nal(2)；其中P2係(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)或(Phe-4CF3)；其中P3係Ser；其中P4係Trp；其中P5係Ser；且其中P6係Bpa或(Ser-Tyr)；或包含表示為P1-P12之殘基之肽，其具有任一以下結構：P1、P2、P3、P4、P5、P6；P6、P5、P4、P3、P2、P1；P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12；P1、P2、P3、P4、P5、P6、P12、P11、P10、P9、P8、P7；P6、P5、P4、P3、P2、P1、P7、P8、P9、P10、P11、P12；P6、P5、P4、P3、P2、P1、P12、P11、P10、P9、 P8、P7；P7、P8、P9、P10、P11、P12、P1、P2、P3、P4、P5、P6；P7、P8、P9、P10、P11、P12、P6、P5、P4、P3、P2、P1；P12、P11、P10、P9、P8、P7、P1、P2、P3、P4、P5、P6；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1；P12、P11、P6、P9、P8、P7、P2、P1；P12、P11、P10、P6、P9、P4、P7、P2、P1；P1、P2、P7、P8、P9、P6、P11、P12；或P1、P2、P7、P4、P9、P6、P10、P11、P12；其中P1係Cha或Nal(2)；其中P2係(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)或(Phe-4CF3)；其中P3係任一胺基酸；其中P4係d-Trp或l-Trp；其中P5係任一胺基酸；其中P6係Bpa或(Ser-Tyr)；其中P7係Arg；其中P8係Arg；其中P9係Arg；其中P10係Gln或Arg；其中P11係Arg；且其中P12係d-Arg或l-Arg，或K)之肽，其中該任一胺基酸係Ser或Pro；或包含表示為P1-P12之殘基之肽，其具有任一以下結構：P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1；P12、P11、P10、P6、P9、P4、P7、P2、P1；或P1、P2、P7、P4、P9、P6、P10、P11、P12；其中P1係Cha或Nal(2)；其中P2係(Phe-2,3,4,5,6-F)；其中P3係Ser；其中P4係Trp；其中P5係Ser；其中P6係Bpa或(Ser-Tyr)；其中P7係Arg；其中P8係Arg；其中P9係Arg；其中P10係Gln或Arg；其中P11係Arg；且其中P12係Arg；或其前藥或其醫藥上可接受之鹽，藉此增加過度增殖細胞之核酸損害或預防或治療細胞增殖病症。 In another embodiment, a method or use for increasing nucleic acid damage in hyperproliferative cells or for preventing or treating a cell proliferative disorder in a mammal (e.g., a human) comprises administering a T cell activator and/or to the mammal or an immune checkpoint inhibitor and a peptide compound, wherein the peptide compound comprises any of the following sequences: A) comprising residues denoted as P1-P6 and having the structure P1, P2, P3, P4, P5, P6 or P6 , P5, P4, P3, P2, and P1 peptides; wherein P1 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), ( Phe-4CF3), amino acids occupying similar side chain spaces, or have one or two aromatic, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or pyrimidine groups in the side chain or one Indole, pentalene, indene, naphthalene, benzofuran, benzothiophene, quinoline, indoline,

Any amino acid of 锍, quinoxaline, quinazoline group; wherein P2 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4 ,5F), (Phe-4CF3), Bpa, Phe4NO2, amino acids that occupy similar side chain space, or have one or two aromatics in the side chain, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine , morpholine or pyrimidine group or an indole, pentalene, indene, naphthalene, benzofuran, benzothiophene, quinoline, indoline,

Any amino acid of N, quinoline or quinazoline group; wherein P3, P4, P5 are any amino acid, or one or more of P3, P4, P5 is a simple carbon chain, so that The distance between P2 and P6 is about the same as when each of P3, P4, and P5 is an amino acid; wherein P6 is Bpa, Phe4NO2, any amino acid, and Tyr, any amino acid, and Phe, any amino acid or absent; peptides of B) or A) in which the amino acids with simple carbon chains are 11-aminoundecanoic acid, 10-aminodecanoic acid, 9-aminononanoic acid , 8-aminocaprylic acid, 7-aminoheptanoic acid, 6-aminocaproic acid, or similar structures with one or more unsaturated carbon bonds, and/or any one of the amino acids is Ser, and/or wherein P4 is Trp, and/or wherein the amino acid occupying a similar side chain space is Tyr or Phe; or a peptide comprising residues denoted P1-P12 having any of the following structures: P1, P2, P3, P4, P5, P6; P6, P5, P4, P3, P2, P1; P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12; P1, P2, P3, P4, P5, P6, P12, P11, P10, P9, P8, P7; P6, P5, P4, P3, P2, P1, P7, P8, P9, P10, P11, P12; P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8, P7; P7, P8, P9, P10, P11, P12, P1, P2, P3, P4, P5, P6; P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1; P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6; P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P6, P9, P8, P7, P2, P1; P12, P11, P10, P6, P9, P4, P7, P2, P1; P1, P2, P7, P8, P9, P6, P11, P12; or P1, P2, P7, P4, P9, P6, P10, P11, P12; where P1 is Cha, Nal(2), (Phe-2,3,4,5, 6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, amino acids occupying similar side chain spaces (such as d-Tyr or l-Tyr, d-Phe or l- Phe), or with one or two aromatic, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or pyrimidine groups in the side chain or an indole, pentalene, indene, naphthalene , Benzofuran, Benzothiophene, Quinoline, Indoline,

Any amino acid of 锍, quinoxaline or quinazoline group; wherein P2 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4 ,5F), (Phe-4CF3) or amino acids occupying similar side chain spaces, or have one or two aromatic, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or a pyrimidine group or an indole, pentalene, indene, naphthalene, benzofuran, benzothiophene, quinoline, indoline,

Any amino acid of alkane, quinoxaline, quinazoline group; wherein P3, P4, P5 are any amino acid, or one or more of P3, P4, P5 are simple carbon chains, so that The distance between P2 and P6 is about the same as when each of P3, P4, and P5 is an amino acid; wherein P6 is Bpa, Phe4NO2, any amino acid, and Tyr, any amino acid, and Phe; and wherein at least three of P7, P8, P9, P10, P11, and P12 are basic amino acids, and the rest are any amino acids or do not exist; or C) a peptide with a simple carbon chain The amino acids are 11-aminoundecanoic acid, 10-aminodecanoic acid, 9-aminononanoic acid, 8-aminooctanoic acid, 7-aminoheptanoic acid, 6-aminocaproic acid or have one or A similar structure of multiple unsaturated carbon bonds, and/or, wherein any one of the amino acids is Ser, and/or, wherein P4 is Trp, and/or, wherein the amino acid occupying a similar side chain space is Tyr or Phe; or a peptide comprising residues denoted P1-P12 having any of the following structures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12; P12, P11 , P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P10, P6, P9, P4, P7, P2, P1; or P1, P2, P7, P4, P9, P6, P10, P11, P12; where P1 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, amino acids occupying similar side chain spaces, or having one or two aromatic, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or pyrimidine groups in the side chain or an indole Indole, pentadiene, indene, naphthalene, benzofuran, benzothiophene, quinoline, indoline,

Any amino acid of 锍, quinoxaline or quinazoline group; wherein P2 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4 ,5F), (Phe-4CF3), amino acids that occupy similar side chain spaces, or have one or two aromatics in the side chain, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or a pyrimidine group or an indole, pentalene, indene, naphthalene, benzofuran, benzothiophene, quinoline, indoline,

Any amino acid of N, quinoline, quinazoline group; wherein P3, P4, P5 are any amino acid, or one or more of P3, P4, P5 is a simple carbon chain, so that The distance between P2 and P6 is about the same as when each of P3, P4, and P5 is an amino acid; wherein P6 is Bpa, Phe4NO2, any amino acid, and Tyr, any amino acid, and Phe, any amino acid or does not exist; and wherein at least three of P7, P8, P9, P10, P11, P12 are basic amino acids, and the rest are any amino acids or do not exist; or E ), wherein the amino acid with a simple carbon chain is aminoundecanoic acid or 8-aminooctanoic acid, and/or, wherein any one of the amino acids is Ser, and/or, wherein it occupies a similar side chain space The amino acid is Tyr or Phe; or a peptide comprising residues denoted P1-P12, which has any of the following structures: P1, P2, P3, P4, P5, P6 or P6, P5, P4, P3, P2, P1, where P1 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, Tyr or Phe; where P2 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, Tyr or Phe; where P3 is Ser, Arg, Cys, Pro or Asn; where P4 is Trp; where P5 is Ser, Arg or Asn; or where P3, P4, P5 is a single amino undecanoic acid or a single 8-amine and wherein P6 is Bpa, Phe4NO2, (Ser-Tyr) or (Ser-Phe); or a peptide comprising residues represented as P1-P12, which has any of the following structures: P1, P2, P3, P4 , P5, P6, P7, P8, P9, P10, P11, P12; P1, P2, P3, P4, P5, P6, P12, P11, P10, P9, P8, P7; P6, P5, P4, P3, P2 , P1, P7, P8, P9, P10, P11, P12; P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8, P7; P7, P8, P9, P10, P11, P12 , P1, P2, P3, P4, P5, P6; P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1; P12, P11, P10, P9, P8, P7, P1 , P2, P3, P4, P5, P6; P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P6, P9, P8, P7, P2, P1 ; P12, P11, P10, P6, P9, P4, P7, P2, P1; P1, P2, P7, P8, P9, P6, P11, P12; or P1, P2, P7, P4, P9, P6, P10, P11, P12; where P1 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, Tyr or Phe; where P2 is Cha, Nal(2), (Phe-2,3,4,5,6-F), (Phe-3,4,5F), (Phe-4CF3), Bpa, Phe4NO2, Tyr or Phe; where P3 is Ser, Arg, Cys, Pro or Asn; where P4 is Trp; where P5 is Ser, Arg or Asn; or where P3, P4, P5 is a single amino undecanoic acid or a single 8-amine Octanoic acid; wherein P6 is Bpa, Phe4NO2, (d-Ser-d-Tyr) or (d-Ser-d-Phe); and wherein at least three of P7, P8, P9, P10, P11, and P12 are Arg or Lys, and the remainder is either amino acid or absent; or a peptide comprising residues denoted P1-P12 having any of the following structures: P1, P2, P3, P4, P5, P6, P7, P8 , P9, P10, P11, P12; P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P10, P6, P9, P4, P7, P2, P1 or P1, P2, P7, P4, P9, P6, P10, P11, P12; wherein P1 is Cha or Nal(2); wherein P2 is (Phe-2,3,4,5,6-F), ( Phe-3,4,5F), (Phe-4CF3); where P3 is Ser; where P4 is Trp; where P5 is Ser or Asn; where P6 is Bpa, Phe4NO2, (Ser-Tyr) or (Ser-Phe) and wherein at least three of P7, P8, P9, P10, P11, P12 are Arg, and the rest are any amino acid or do not exist; or a peptide comprising residues represented as P1-P12, which has any - The following structures: P1, P2, P3, P4, P5, P6 or P6, P5, P4, P3, P2, P1; where P1 is Cha or Nal(2); where P2 is (Phe-2,3,4, 5,6-F), (Phe-3,4,5F) or (Phe-4CF3); wherein P3 is Ser; wherein P4 is Trp; wherein P5 is Ser; and wherein P6 is Bpa or (Ser-Tyr); Or a peptide comprising residues denoted P1-P12, which has any of the following structures: P1, P2, P3, P4, P5, P6; P6, P5, P4, P3, P2, P1; P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12; P1, P2, P3, P4, P5, P6, P12, P11, P10, P9, P8, P7; P6, P5, P4, P3, P2, P1, P7, P8, P9, P10, P11, P12; P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8, P7; P7, P8, P9, P10, P11, P12, P1, P2, P3, P4, P5, P6; P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1; P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6; P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1; P12, P11, P6, P9, P8, P7, P2, P1; P12, P11, P10, P6, P9, P4, P7, P2, P1; P1, P2, P7, P8, P9, P6, P11, P12; or P1, P2, P7, P4, P9, P6, P10 , P11, P12; where P1 is Cha or Nal(2); where P2 is (Phe-2,3,4,5,6-F), (Phe-3,4,5F) or (Phe-4CF3); wherein P3 is any amino acid; wherein P4 is d-Trp or l-Trp; wherein P5 is any amino acid; wherein P6 is Bpa or (Ser-Tyr); wherein P7 is Arg; wherein P8 is Arg; wherein P9 is Arg; wherein P10 is Gln or Arg; wherein P11 is Arg; and wherein P12 is d-Arg or 1-Arg, or K), wherein any amino acid is Ser or Pro; A peptide that is the residues of P1-P12, which has any of the following structures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12; P12, P11, P10, P9, P8 , P7, P6, P5, P4, P3, P2, P1; P12, P11, P10, P6, P9, P4, P7, P2, P1; or P1, P2, P7, P4, P9, P6, P10, P11, P12; where P1 is Cha or Nal(2); where P2 is (Phe-2,3,4,5,6-F); where P3 is Ser; where P4 is Trp; where P5 is Ser; where P6 is Bpa or (Ser-Tyr); wherein P7 is Arg; wherein P8 is Arg; wherein P9 is Arg; wherein P10 is Gln or Arg; wherein P11 is Arg; and wherein P12 is Arg; or a prodrug thereof or pharmaceutically acceptable thereof salts, thereby increasing nucleic acid damage in hyperproliferative cells or preventing or treating cell proliferative disorders.

在特定實施例中，方法或用途採用包括任一以下序列之肽化合物：(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg) (d-Arg)(d-Gln)(d-Arg)(d-Arg)；(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)；(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)；(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)；(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)；(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)；(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)；(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)；(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)； (d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)；(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)；(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Arg)；(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)；或(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)。 In a particular embodiment, the method or use employs a peptide compound comprising any of the following sequences: (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4, 5,6-F)(d-Cha)(d-Arg)(d-Arg) (d-Arg)(d-Gln)(d-Arg)(d-Arg); (d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg )(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha); (d-Bpa)( d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Gln )(d-Arg)(d-Arg)(d-Arg); (d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d- Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha); (d-Cha)(d-Phe- 2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d- Gln)(d-Arg)(d-Arg); (d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Cha)(d -Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa); (d-Cha)(d-Phe-2,3, 4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d -Arg)(d-Arg); (d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2 ,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa); (d-Arg)(d-Arg)(d-Arg)(d- Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)( d-Bpa); (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d- Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg); (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d -Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) ;(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d -Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg); (d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d- Cha); (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg) (d-Arg); (d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3, 4,5,6-F)(d-Cha); (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg )(d-Bpa)(d-Arg)(d-Arg)(d-Arg); (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d- Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha); (d-Cha)(d-Phe-2,3,4 ,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Arg);(d -Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F) (d-Cha); or (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)( d-Arg)(d-Arg)(d-Arg).

在其他具體實施例中，方法或用途採用包括任一以下序列或由其組成之肽化合物：(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：1)；(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)或(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)。 In other embodiments, the method or use employs a peptide compound comprising or consisting of any of the following sequences: (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2 ,3,4,5,6-F)(d-Cha)(SEQ ID NO: 1 ); (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2 ,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) or (d- Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d- Phe-2,3,4,5,6-F)(d-Cha).

在再具體實施例中，方法或用途採用T細胞活化劑。T細胞活化劑之非限制性實例包括靶向CD28(細胞分化抗原28，亦稱為Tp44、T細胞特異性表面醣蛋白、CD28抗原、CD28分子)、OX40(腫瘤壞死因子受體超家族成員4、TNFRSF4，亦稱為OX40L受體、OX40抗原、TXGP1L)、GITR(糖皮質激素誘導之腫瘤壞死因子受體)、CD137(亦稱為4-1BB)、CD27(亦稱為TNFRSF7、Tp55)及HVEM(皰疹病毒侵入介體，亦稱為CD270、TNFRSF14)之藥劑。 In yet specific embodiments, the method or use employs a T cell activator. Non-limiting examples of T cell activators include targeting CD28 (cell differentiation antigen 28, also known as Tp44, T cell specific Surface glycoprotein, CD28 antigen, CD28 molecule), OX40 (tumor necrosis factor receptor superfamily member 4, TNFRSF4, also known as OX40L receptor, OX40 antigen, TXGP1L), GITR (glucocorticoid-induced tumor necrosis factor receptor ), CD137 (also known as 4-1BB), CD27 (also known as TNFRSF7, Tp55) and HVEM (herpes virus entry mediator, also known as CD270, TNFRSF14).

代表性T細胞活化劑包括結合至該等靶標之配體，例如，CD28、OX40、GITR、CD137、CD27及HVEM配體。代表性T細胞活化劑亦包括結合至該等靶標之抗體，例如，抗CD28、抗OX40、抗GITR、抗CD137、抗CD27及抗HVEM抗體。 Representative T cell activators include ligands that bind to these targets, eg, CD28, OX40, GITR, CD137, CD27, and HVEM ligands. Representative T cell activators also include antibodies that bind to these targets, eg, anti-CD28, anti-OX40, anti-GITR, anti-CD137, anti-CD27, and anti-HVEM antibodies.

在再其他具體實施例中，方法或用途採用免疫查核點抑制劑。免疫查核點抑制劑之非限制性實例包括靶向CTLA-4(細胞毒性T淋巴球相關性蛋白質4，亦稱為CD152)、PD1(程式性細胞死亡1，亦稱為CD279、SLEB2、HPD-1、HSLE1)、PD-L1(程式化死亡-配體1，亦稱為CD274、B7-H1(B7同系物1)、程式性細胞死亡1配體1、PDCD1配體1)、PDL2(程式性細胞死亡1配體2)、VISTA(T細胞活化之V結構域Ig抑制劑，亦稱為B7-H5、Gi24、Dies1及SISP1)、TIM3(T細胞免疫球蛋白及黏蛋白結構域3)、LAG-3(淋巴球活化基因3，亦稱為CD223)或BTLA(B-淋巴球及T-淋巴球弱化子，亦稱為CD272)之藥劑。 In still other specific embodiments, the method or use employs an immune checkpoint inhibitor. Non-limiting examples of immune checkpoint inhibitors include targeting CTLA-4 (cytotoxic T-lymphocyte-associated protein 4, also known as CD152), PD1 (programmed cell death 1, also known as CD279, SLEB2, HPD- 1, HSLE1), PD-L1 (programmed death-ligand 1, also known as CD274, B7-H1 (B7 homologue 1), programmed cell death 1 ligand 1, PDCD1 ligand 1), PDL2 (programmed cell death 1 ligand 2), VISTA (V domain Ig inhibitor of T cell activation, also known as B7-H5, Gi24, Dies1 and SISP1), TIM3 (T cell immunoglobulin and mucin domain 3) , LAG-3 (lymphocyte activation gene 3, also known as CD223) or BTLA (B-lymphocyte and T-lymphocyte attenuator, also known as CD272) agents.

代表性免疫查核點抑制劑包括結合至該等靶標之配體，例如，CTLA-4、PD1、PD-L1、PDL2、VISTA、TIM3、LAG-3及BTLA配體。代表性免疫查核點抑制劑亦包括結合至該等靶標之抗體，例如，抗CTLA-4、抗PD1、抗PD-L1、抗PDL2、抗VISTA、抗TIM3、抗LAG-3及抗BTLA抗體。 Representative immune checkpoint inhibitors include ligands that bind to these targets, eg, CTLA-4, PD1, PD-L1, PDL2, VISTA, TIM3, LAG-3, and BTLA ligands. Representative immune checkpoint inhibitors also include antibodies that bind to these targets, eg, anti-CTLA-4, anti-PDl, anti-PD-Ll, anti-PDL2, anti-VISTA, anti-TIM3, anti-LAG-3, and anti-BTLA antibodies.

在其他具體實施例中，方法或用途係在以下哺乳動物上實踐：具有在正常範圍內之白血球計數之哺乳動物；具有小於約11,000個白血球/微升(wbc/μl)血液之白血球計數之哺乳動物；具有介於約4,000個至約11,000個白血球/微升(wbc/μl)血液之間之白血球計數之哺乳動物；具有小於約10,000個白血球/微升(wbc/μl)血液之白血球計數之哺乳動物；具有小於約9,000個白血球/微升(wbc/μl)血液之白血球計數之哺乳動物；具有介於約4,000個至約9,000個白血球/微升(wbc/μl)血液之間之白血球計數之哺乳動物；具有小於約8,000個白血球/微升(wbc/μl)血液之白血球計數之哺乳動物；具有小於約7,000個白血球/微升(wbc/μl)血液之白血球計數之哺乳動物；或具有小於每一臨床實驗室白血球/微升(wbc/μl)血液之正常值上限之白血球計數之哺乳動物。 In other embodiments, the method or use is practiced on a mammal having a white blood cell count within the normal range; a mammal having a white blood cell count of less than about 11,000 white blood cells per microliter (wbc/μl) of blood Animals; mammals having a white blood cell count between about 4,000 and about 11,000 white blood cells per microliter (wbc/μl) of blood; those having a white blood cell count of less than about 10,000 white blood cells per microliter (wbc/μl) of blood Mammals; mammals having a white blood cell count of less than about 9,000 white blood cells per microliter (wbc/μl) of blood; mammals having a white blood cell count of between about 4,000 to about 9,000 white blood cells per microliter (wbc/μl) of blood mammals; mammals with a white blood cell count of less than about 8,000 white blood cells per microliter (wbc/μl) of blood; mammals with a white blood cell count of less than about 7,000 white blood cells per microliter (wbc/μl) of blood; or mammals with Mammals with white blood cell counts less than the upper limit of normal for white blood cells per microliter (wbc/μl) of blood per clinical laboratory.

方法及用途在醫藥調配物中包括肽化合物。方法及用途亦在醫藥調配物中包括T細胞活化劑及/或免疫查核點抑制劑。本發明方法及用途亦包括藉由任一途徑投與。在特定實施例中，肽化合物係以局部、區域性或全身方式投與。 Methods and Uses Peptide compounds are included in pharmaceutical formulations. Methods and Uses Also include T cell activators and/or immune checkpoint inhibitors in pharmaceutical formulations. The methods and uses of the present invention also include administration by any route. In particular embodiments, the peptide compounds are administered locally, regionally or systemically.

方法及用途包括肽化合物之醫藥上可接受之鹽。在具體態樣中，醫藥上可接受之鹽係以下各項中之任一者或其組合：乙酸鹽、磺酸鹽、硫酸鹽、焦硫酸鹽、硫酸氫鹽、亞硫酸鹽、亞硫酸氫鹽、磷酸鹽、磷酸氫鹽、磷酸二氫鹽、偏磷酸鹽、焦磷酸鹽、氯化物、溴化物、碘化物、丙酸鹽、癸酸鹽、辛酸鹽、丙烯酸鹽、甲酸鹽、異丁酸鹽、己酸鹽、庚酸鹽、丙炔酸鹽、草酸鹽、丙二酸鹽、琥珀酸鹽、辛二酸鹽、癸二酸鹽、富馬酸鹽、馬來酸鹽、丁炔-1,4-二酸鹽、己炔-1,6-二酸鹽、苯甲酸鹽、氯苯甲酸鹽、苯甲酸甲酯、二硝基苯甲酸鹽、羥基苯甲酸鹽、甲氧基苯甲酸鹽、鄰苯二甲酸鹽、二甲苯磺酸鹽、苯基乙酸鹽、苯基丙酸鹽、苯基丁酸鹽、檸檬酸鹽、乳酸鹽、γ-羥基丁酸鹽、羥乙酸鹽、酒石酸鹽、甲烷-磺酸鹽、丙烷磺酸鹽、萘-1-磺酸鹽、萘-2-磺酸鹽及杏仁酸鹽。 Methods and uses include pharmaceutically acceptable salts of peptide compounds. In specific aspects, the pharmaceutically acceptable salt is any one or combination of the following: acetate, sulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite Salt, phosphate, hydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, propionate, caprate, caprylate, acrylate, formate, iso Butyrate, Caproate, Heptanoate, Propiolate, Oxalate, Malonate, Succinate, Suberate, Sebacate, Fumarate, Maleate, Butyne-1,4-dioate, Hexyne-1,6-dioate, Benzoate, Chlorobenzoate, Methyl Benzoate, Dinitrobenzoate, Hydroxybenzoic Acid Salt, Methoxybenzoates, Phthalates, Xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methane-sulfonate, Propane sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate.

方法及用途包括肽化合物包括6至10個、10至15個、15至20個、20至25個、25至30個、30至40個、40至50個、50至75個、75至100個、100至150個、150至200個或200至300個胺基酸殘基長度或由其組成。 Methods and uses include peptide compounds including 6 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100 , 100 to 150, 150 to 200, or 200 to 300 amino acid residues in length or consist thereof.

方法及用途包括肽化合物包括與其附接或偶聯之細胞穿透分子或由其組成。在具體非限制性態樣中，細胞穿透分子係藉由共價鍵或肽或非肽連接體接合至肽化合物。在其他具體非限制性態樣中，細胞穿透肽包含極性/帶電胺基酸及非極性、疏水胺基酸之替代型式。在再其他具體非限制性態樣中，細胞穿透肽包含多陽離子或兩親性α-螺旋結構。在再其他具體非限制性態樣中，細胞穿透肽包含聚-精胺酸(Arg)序列(例如，肽包括(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)或由其組成)。 Methods and uses include peptide compounds comprising or consisting of cell penetrating molecules attached or coupled thereto. In a specific non-limiting aspect, the cell penetrating molecule is joined to the peptidic compound by a covalent bond or a peptide or non-peptide linker. In other specific, non-limiting aspects, the cell penetrating peptides comprise polar/charged amino acids and alternative versions of non-polar, hydrophobic amino acids. In still other specific non-limiting aspects, the cell penetrating peptide comprises a polycation or an amphipathic alpha-helical structure. In still other specific non-limiting aspects, the cell penetrating peptide comprises a poly-arginine (Arg) sequence (e.g., the peptide comprises (d-Arg)(d-Arg)(d-Arg)(d-Gln) (d-Arg)(d-Arg) or consist thereof).

在再其他具體態樣中，肽化合物及/或細胞穿透肽及/或T細胞活化劑及/或免疫查核點抑制劑包括L-異構物或D-異構物胺基酸或L-異構物及D-異構物胺基酸之混合物或由其組成。 In yet other specific aspects, the peptidic compound and/or cell penetrating peptide and/or T cell activator and/or immune checkpoint inhibitor comprises an L-isomer or a D-isomer amino acid or an L- A mixture of or consisting of isomer and D-isomer amino acids.

在其他具體實施例中，方法或用途包括投與T細胞活化劑及肽化合物。在其他具體實施例中，方法或用途包括投與免疫查核點抑制劑及肽化合物。在其他具體實施例中，方法或用途包括投與T細胞活化劑及免疫查核點抑制劑及肽化合物。 In other embodiments, the method or use comprises administering a T cell activator and a peptide compound. In other embodiments, the method or use comprises administering an immune checkpoint inhibitor and a peptide compound. In other embodiments, the methods or uses comprise administering T cell activators and immune checkpoint inhibitors and peptide compounds.

在其他具體實施例中，方法或用途包括投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療。非限制性核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療包括手術切除、放射療法、離子化或化學放射療法、化學療法、免疫療法、局部或區域性熱(發熱)療法、疫苗接種、烷基化劑、抗代謝物、植物提取物、植物鹼、亞硝基脲、激素或核苷或核苷酸類似物或由其組成。 In other embodiments, the method or use comprises administering a nucleic acid damaging agent, nucleic acid damaging therapy, antiproliferative agent or antiproliferative therapy. Non-limiting nucleic acid damaging agents, nucleic acid damaging treatments, antiproliferative agents or antiproliferative treatments include surgical resection, radiation therapy, ionizing or chemoradiation therapy, chemotherapy chemotherapy, immunotherapy, local or regional heat (fever) therapy, vaccination, alkylating agents, antimetabolites, plant extracts, plant alkaloids, nitrosoureas, hormones, or nucleoside or nucleotide analogs or consisting of it.

在再其他具體實施例中，方法或用途進一步包括在投與T細胞活化劑之前、與此同時或在此之後投與肽化合物或由其組成。在再其他具體實施例中，方法或用途進一步包括在投與免疫查核點抑制劑之前、與此同時或在此之後投與肽化合物或由其組成。在再其他具體實施例中，方法或用途進一步包括與T細胞活化劑組合投與肽化合物或由其組成。在再其他具體實施例中，方法或用途進一步包括與免疫查核點抑制劑組合投與肽化合物或由其組成。在再其他具體實施例中，方法或用途進一步包括與T細胞活化劑及免疫查核點抑制劑組合投與肽化合物或由其組成。 In yet other specific embodiments, the method or use further comprises administering, or consisting of, the peptide compound prior to, concurrently with, or subsequent to administering the T cell activator. In yet other embodiments, the method or use further comprises administering, or consisting of, the peptide compound prior to, concurrently with, or subsequent to the administration of the immune checkpoint inhibitor. In still other specific embodiments, the method or use further comprises or consists of administering the peptide compound in combination with a T cell activator. In yet other specific embodiments, the method or use further comprises or consists of administering the peptide compound in combination with an immune checkpoint inhibitor. In still other embodiments, the methods or uses further comprise or consist of administering the peptide compound in combination with a T cell activator and an immune checkpoint inhibitor.

在具體態樣中，肽化合物係在投與T細胞活化劑及/或免疫查核點抑制劑之前或之後小於48小時投與；肽化合物係在投與T細胞活化劑及/或免疫查核點抑制劑之前或之後不到24小時投與；肽化合物係在投與T細胞活化劑及/或免疫查核點抑制劑之前或之後不到12小時投與；肽化合物係在投與T細胞活化劑及/或免疫查核點抑制劑之前或之後不到6小時投與；肽化合物係在投與T細胞活化劑及/或免疫查核點抑制劑之前或之後不到4小時投與，肽化合物係在投與T細胞活化劑及/或免疫查核點抑制劑之前或之後不到2小時投與；肽化合物係在投與T細胞活化劑及/或免疫查核點抑制劑之前或之後不到1小時投與。 In specific aspects, the peptide compound is administered less than 48 hours before or after administration of the T cell activator and/or immune checkpoint inhibitor; the peptide compound is administered after the administration of the T cell activator and/or immune checkpoint inhibitor The peptide compound is administered less than 24 hours before or after the administration of the T cell activator and/or immune checkpoint inhibitor; The peptide compound is administered less than 12 hours after the administration of the T cell activator and/or immune checkpoint inhibitor; The peptide compound is administered less than 4 hours before or after the T cell activator and/or the immune checkpoint inhibitor, and the peptide compound is administered less than 6 hours before or after the immune checkpoint inhibitor. Administered less than 2 hours before or after T cell activators and/or immune checkpoint inhibitors; peptide compound is administered less than 1 hour before or after T cell activators and/or immune checkpoint inhibitors .

在再其他具體實施例中，方法或用途進一步包括在投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療之前、與此同時或在此之後投與肽化合物、T細胞活化劑及/或免疫查核點抑制劑或由其組成。在具體態樣中，肽化合物、T細胞活化劑及/或免疫查核點抑制劑係在投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療之前或之後不到48小時投與；肽化合物、T細胞活化劑及/或免疫查核點抑制劑係在投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療之前或之後不到24小時投與；肽化合物、T細胞活化劑及/或免疫查核點抑制劑係在投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療之前或之後不到12小時投與；肽化合物、T細胞活化劑及/或免疫查核點抑制劑係在投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療之前或之後不到6小時投與；肽化合物、T細胞活化劑及/或免疫查核點抑制劑係在投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療之前或之後不到4小時投與；肽化合物、T細胞活化劑及/或免疫查核點抑制劑係在投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療之前或之後不到2小時投與；肽化合物、T細胞活化劑及/或免疫查核點抑制劑係在投與核酸損害劑、核酸損害治療、抗增殖劑或抗增殖治療之前或之後不到1小時投與。 In still other specific embodiments, the method or use further comprises administering the peptide compound, T cell activator, and / or an immune checkpoint inhibitor or consisting of it. In a specific form, The peptide compound, T cell activator, and/or immune checkpoint inhibitor is administered less than 48 hours before or after administration of a nucleic acid damaging agent, nucleic acid damaging therapy, antiproliferative agent, or antiproliferative therapy; peptide compound, T cell activation The agent and/or immune checkpoint inhibitor is administered less than 24 hours before or after administration of nucleic acid damaging agent, nucleic acid damaging therapy, antiproliferative agent or antiproliferative therapy; peptide compound, T cell activator and/or immune checkpoint The point inhibitor is administered before or less than 12 hours after administration of the nucleic acid damaging agent, nucleic acid damaging therapy, antiproliferative agent, or antiproliferative therapy; the peptide compound, T cell activator, and/or immune checkpoint inhibitor is administered Administered less than 6 hours before or after nucleic acid damaging agents, nucleic acid damaging therapies, antiproliferative agents, or antiproliferative therapies; peptide compounds, T cell activators, and/or immune checkpoint inhibitors were administered before nucleic acid damaging agents, nucleic acid Administered before or less than 4 hours after damaging therapy, antiproliferative agent, or antiproliferative therapy; peptide compound, T cell activator, and/or immune checkpoint inhibitor is administered before nucleic acid damaging agent, nucleic acid damaging therapy, antiproliferative agent or less than 2 hours before or after antiproliferative therapy; peptide compound, T cell activator and/or immune checkpoint inhibitor is administered before nucleic acid damaging agent, nucleic acid damaging therapy, antiproliferative agent or antiproliferative therapy or Dose less than 1 hour later.

核酸損害劑或抗增殖劑之非限制性實例包括藥物。核酸損害劑或抗增殖劑之非限制性實例包括含鉑藥物，例如順鉑(cis-platin)、卡鉑(carboplatin)、奈達鉑(nedaplatin)、米他鉑(mitaplatin)、沙鉑(satraplatin)、匹鉑(picoplatin)、三鉑(triplatin)、米利鉑(miriplatin)或奧沙利鉑(oxaliplatin)。 Non-limiting examples of nucleic acid damaging or antiproliferative agents include drugs. Non-limiting examples of nucleic acid damaging or antiproliferative agents include platinum-containing drugs such as cis-platin, carboplatin, nedaplatin, mitaplatin, satraplatin ), picoplatin, triplatin, miriplatin, or oxaliplatin.

更具體而言，方法及用途包括投與以下各項或由其組成：含鉑藥物順鉑、卡鉑、奧沙利鉑、培美曲塞(pemetrexed)、吉西他濱(gemcitabine)、5-氟尿嘧啶(5-FU)、蝴蝶黴素(rebeccamycin)、阿德力黴素(adriamycin，ADR)、博來黴素(bleomycin，Bleo)、派來黴素(pepleomycin)、順鉑、順鉑或順式-二氯二胺鉑(II)(CDDP)、奧沙利鉑或喜樹鹼(camptothecin，CPT)、環磷醯胺、硫唑嘌呤、環孢素A(cyclosporin A)、普賴蘇濃(prednisolone)、美法侖(melphalan)、氮芥苯丁酸(chlorambucil)、甲基二(氯乙基)胺、白消安(busulphan)、胺甲喋呤(methotrexate)、6-巰嘌呤、硫鳥嘌呤、5-氟尿嘧啶、胞嘧啶阿拉伯糖苷(cytosine arabinoside)、AZT、5-氮胞苷(5-AZC)或5-氮胞苷相關化合物、放線菌素D(actinomycin D)、光輝黴素(mithramycin)、絲裂黴素C(mitomycin C)、卡莫司汀(carmustine)、洛莫司汀(lomustine)、司莫司汀(semustine)、鏈佐黴素(streptozotocin)、羥基脲、順鉑、米托坦(mitotane)、丙卡巴肼(procarbazine)、達卡巴嗪(dacarbazine)、紫杉烷(taxane)、長春鹼(vinblastine)、長春新鹼(vincristine)、多柔比星(doxorubicin)、二溴甘露醇、輻射或放射性同位素。輻射之具體非限制性實例包括UV輻射、IR輻射、X射線或α輻射、β輻射或γ-輻射。放射性同位素之具體非限制性實例包括I¹³¹、I¹²⁵、Sr⁸⁹、Sm¹⁵³、Y⁹⁰或Lu¹⁷⁷。 More specifically, the methods and uses include administering or consisting of the following: the platinum-containing drugs cisplatin, carboplatin, oxaliplatin, pemetrexed, gemcitabine, 5-fluorouracil ( 5-FU), rebeccamycin, adriamycin (ADR), bleomycin (Bleo), pepleomycin, cisplatin, cisplatin or cis- Platinum(II) dichlorodiamine (CDDP), oxaliplatin or camptothecin (CPT), cyclophosphamide, azathioprine, cyclosporin A, prednisolone ), melphalan, chlorambucil, methylbis(chloroethyl)amine, busulphan, methotrexate, 6-mercaptopurine, thioguanine Purine, 5-fluorouracil, cytosine arabinoside (cytosine arabinoside), AZT, 5-azacytidine (5-AZC) or 5-azacytidine related compounds, actinomycin D, mithramycin ), mitomycin C, carmustine, lomustine, semustine, streptozotocin, hydroxyurea, cisplatin, Mitotane, procarbazine, dacarbazine, taxane, vinblastine, vincristine, doxorubicin, Bromomannitol, radiation or radioisotopes. Specific non-limiting examples of radiation include UV radiation, IR radiation, X-rays or alpha, beta or gamma radiation. Specific non-limiting examples of radioactive isotopes include I ¹³¹ , I ¹²⁵ , Sr ⁸⁹ , Sm ¹⁵³ , Y ⁹⁰ , or Lu ¹⁷⁷ .

本發明方法及用途可應用於細胞增殖或過度增殖病症或不合意之細胞增殖。在具體實施例中，細胞增殖病症包含腫瘤或癌症。在更具體實施例中，細胞增殖病症包含轉移性腫瘤或癌症。 The methods and uses of the invention are applicable to cell proliferation or hyperproliferative disorders or undesired cell proliferation. In specific embodiments, the cell proliferative disorder comprises a tumor or cancer. In more specific embodiments, the cell proliferative disorder comprises metastatic tumor or cancer.

腫瘤或癌症之具體非限制性實例包括肺腫瘤或癌症，例如小細胞或非小細胞肺癌或腺癌、鱗狀細胞癌或大細胞癌。腫瘤或癌症之其他具體非限制性實例包括癌、肉瘤、淋巴瘤、白血病、腺瘤、腺癌、黑色素瘤、神經膠質瘤、神經膠母細胞瘤、腦脊髓膜瘤、神經胚細胞瘤、視網膜母細胞瘤、星細胞瘤、少突膠質細胞瘤、間皮瘤、網狀內皮、淋巴或造血性贅瘤形成、腫瘤、癌症或惡性病。腫瘤或癌症之其他具體非限制性實例係肺、甲狀腺、頭或頸、鼻咽、喉、鼻或鼻竇、腦、脊柱、乳房、腎上腺、垂體、甲狀腺、淋巴、胃腸(口腔、食管、胃、十二指腸、迴腸、空腸(小腸)、結腸、直腸)、生殖泌尿道(子宮、卵巢、子宮頸、子宮內膜、膀胱、睪丸、陰莖、前列腺)、腎、胰臟、肝、骨、骨髓、淋巴、血液、肌肉或皮膚之贅瘤形成、腫瘤或癌症。腫瘤或癌症之再其他具體非限制性實例包括乳癌、前列腺癌、胰臟癌、胃癌、胸膜間皮瘤、結腸癌、直腸癌、大腸癌、小腸癌、食管癌、十二指腸癌、舌癌、咽癌、唾液腺癌、腦瘤、神經鞘瘤、肝癌、腎癌、膽管癌、子宮內膜癌、子宮頸癌、子宮體癌、卵巢癌、膀胱癌、尿道癌、皮膚癌、血管瘤、惡性淋巴瘤、惡性黑色素瘤、甲狀腺癌、副甲狀腺癌、鼻癌、鼻竇癌、聽覺器官癌、口底癌、喉癌、腮腺癌、頜下癌、骨腫瘤、血管纖維瘤、視網膜肉瘤、陰莖癌、睪丸腫瘤、小兒實體癌、卡波西氏肉瘤、上頜竇腫瘤、纖維性組織細胞瘤、平滑肌肉瘤、橫紋肌肉瘤、淋巴瘤、多發性骨髓瘤或白血病。 Specific non-limiting examples of tumors or cancers include lung tumors or cancers, such as small cell or non-small cell lung cancer or adenocarcinoma, squamous cell carcinoma or large cell carcinoma. Other specific non-limiting examples of tumors or cancers include carcinoma, sarcoma, lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma, retinal Blastoma, astrocytoma, oligodendroglioma, mesothelioma, reticuloendothelial, lymphoid or hematopoietic neoplasia, neoplasm, cancer or malignancy. Other specific non-limiting examples of tumors or cancers are lung, thyroid, Head or neck, nasopharynx, larynx, nose or sinuses, brain, spine, breast, adrenal, pituitary, thyroid, lymphatic, gastrointestinal (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), reproductive Neoplasia, tumor or cancer of urinary tract (uterus, ovary, cervix, endometrium, bladder, testis, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood, muscle or skin. Still other specific non-limiting examples of tumors or cancers include breast cancer, prostate cancer, pancreatic cancer, gastric cancer, pleural mesothelioma, colon cancer, rectal cancer, large intestine cancer, small intestine cancer, esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer Carcinoma, salivary gland cancer, brain tumor, schwannoma, liver cancer, kidney cancer, bile duct cancer, endometrial cancer, cervical cancer, uterine body cancer, ovarian cancer, bladder cancer, urethral cancer, skin cancer, hemangioma, malignant lymphoma tumor, malignant melanoma, thyroid cancer, parathyroid cancer, nasal cancer, sinus cancer, auditory organ cancer, mouth floor cancer, laryngeal cancer, parotid gland cancer, submandibular cancer, bone tumor, angiofibroma, retinosarcoma, penile cancer, Testicular tumor, pediatric solid cancer, Kaposi's sarcoma, maxillary sinus tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, lymphoma, multiple myeloma, or leukemia.

肉瘤之具體非限制性實例包括淋巴肉瘤、脂肪肉瘤、骨肉瘤、軟骨肉瘤、平滑肌肉瘤、橫紋肌肉瘤或纖維肉瘤。造血性腫瘤、癌症或惡性病之具體非限制性實例包括骨髓瘤、淋巴瘤或白血病。 Specific non-limiting examples of sarcomas include lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma, or fibrosarcoma. Specific non-limiting examples of hematopoietic neoplasms, cancers or malignancies include myeloma, lymphoma or leukemia.

本發明方法及用途包括投與有效治療腫瘤或癌症之量之肽化合物。在具體態樣中，方法或用途抑制或減少腫瘤或癌症之復發、生長、進展、惡化或轉移；使得贅瘤、腫瘤、癌症或惡性細胞團塊、細胞之體積、大小或數量部分地或完全地破壞，刺激、誘導或增加贅瘤、腫瘤、癌症或惡性細胞壞死、細胞溶解或細胞凋亡，減少贅瘤形成、腫瘤、癌症或惡性病體積大小、細胞團塊，抑制或預防贅瘤形成、腫瘤、癌症或惡性病體積、團塊、大小或細胞數量進展或增加或延長壽命；使得減少或降低與贅瘤形成、腫瘤、癌症或惡性病相關或由其造成之不利症狀或併發症之嚴重性、持續時間或頻率；或方法使得減少或降低疼痛、不適、噁心、虛弱或嗜睡，或使得精力、食欲增加，活動力或心理幸福感改良。 The methods and uses of the present invention comprise administering the peptide compound in an amount effective to treat tumor or cancer. In a specific aspect, the method or use inhibits or reduces tumor or cancer recurrence, growth, progression, progression or metastasis; causes neoplasm, tumor, cancer or malignant cell mass, volume, size or number of cells to be partially or completely Destroy, stimulate, induce or increase neoplasia, tumor, cancer or malignant cell necrosis, cell lysis or apoptosis, reduce neoplasia formation, tumor, cancer or malignant disease volume, cell mass, inhibit or prevent neoplasia formation , tumor, cancer or malignancy volume, mass, size or cell number progression or increase or prolong life; severity, duration, or frequency of adverse symptoms or complications associated with or resulting from neoplasms, cancer, or malignancy; or methods that result in less or less pain, discomfort, nausea, weakness, or lethargy, or that increase energy, appetite, activity Improved energy or psychological well-being.

在其他具體態樣中，方法或用途在贅瘤形成、腫瘤、癌症或惡性病中刺激、誘導或增加CD8(=CD45+CD8+)T細胞群體。在贅瘤形成、腫瘤、癌症或惡性病中經刺激、經誘導或有所增加之T細胞群體可為CD8(=CD45+CD8+)T細胞之存活及/或浸潤提高之結果。 In other specific aspects, the method or use stimulates, induces or increases a population of CD8 (=CD45+CD8+) T cells in a neoplasia, tumor, cancer or malignancy. The stimulated, induced or increased T cell population in neoplasia, tumor, cancer or malignancy may be the result of increased survival and/or infiltration of CD8 (=CD45+CD8+) T cells.

在其他具體態樣中，方法或用途在贅瘤形成、腫瘤、癌症或惡性病中減少或降低M2巨噬細胞(=F4/80+CD206+)群體。在贅瘤形成、腫瘤、癌症或惡性病中減少或降低之M2巨噬細胞(=F4/80+CD206+)群體可為M2巨噬細胞(=F4/80+CD206+)之存活及/或浸潤減少之結果。 In other specific aspects, the method or use reduces or reduces the population of M2 macrophages (=F4/80+CD206+) in a neoplasia, tumor, cancer or malignancy. Decreased or decreased M2 macrophage (=F4/80+CD206+) population in neoplasia, tumor, cancer or malignancy may be decreased survival and/or infiltration of M2 macrophages (=F4/80+CD206+) the result.

此外，提供套組，其包括肽化合物、T細胞活化劑及/或免疫查核點抑制劑視情況以及核酸損害治療(例如，核酸損害劑)或抗增殖劑。在一個實施例中，套組包括肽化合物及用於實踐本發明方法之說明書。在另一實施例中，套組包括肽化合物、T細胞活化劑及/或免疫查核點抑制劑及用於實踐本發明方法之說明書。在另一實施例中，套組包括肽化合物、免疫查核點抑制劑及/或T細胞活化劑及用於實踐本發明方法之說明書。在另一實施例中，套組包括肽化合物、T細胞活化劑及/或免疫查核點抑制劑及用於實踐本發明方法之說明書。在再其他實施例中，套組包括肽化合物、T細胞活化劑及免疫查核點抑制劑及用於實踐本發明方法之說明書。 In addition, kits are provided that include a peptide compound, a T cell activator and/or an immune checkpoint inhibitor as appropriate, and a nucleic acid damaging treatment (eg, a nucleic acid damaging agent) or an antiproliferative agent. In one embodiment, a kit includes a peptide compound and instructions for practicing the methods of the invention. In another embodiment, a kit includes a peptide compound, a T cell activator and/or an immune checkpoint inhibitor and instructions for practicing the methods of the invention. In another embodiment, a kit includes a peptide compound, an immune checkpoint inhibitor and/or a T cell activator, and instructions for practicing the methods of the invention. In another embodiment, a kit includes a peptide compound, a T cell activator and/or an immune checkpoint inhibitor and instructions for practicing the methods of the invention. In yet other embodiments, a kit includes a peptide compound, a T cell activator, and an immune checkpoint inhibitor, and instructions for practicing the methods of the invention.

圖1顯示在用於對抗博來黴素處理之Jurkat細胞時每一化合物之劑量反應曲線。X軸指示劑量且Y軸指示治療後之G2/M細胞%。 Figure 1 shows the dose response curves for each compound when used against bleomycin-treated Jurkat cells. X-axis indicates dose and Y-axis indicates % G2/M cells after treatment.

圖2顯示在用於對抗秋水仙鹼處理之Jurkat細胞時每一化合物之劑量反應曲線。X軸指示劑量且Y軸指示治療後之G2/M細胞%。 Figure 2 shows the dose response curves for each compound when used against colchicine-treated Jurkat cells. X-axis indicates dose and Y-axis indicates % G2/M cells after treatment.

圖3A及3B經(A)博來黴素(Bleo)或(B)阿德力黴素(ADR)以及各種劑量之化合物處理之人類胰臟癌源性細胞系MIAPaCa2。對所收穫細胞之DNA進行染色並利用流式細胞術加以分析。sub-G1細胞群體之%指示為死細胞。 Figures 3A and 3B Human pancreatic cancer-derived cell line MIAPaCa2 treated with (A) bleomycin (Bleo) or (B) adriamycin (ADR) and various doses of compounds. DNA from harvested cells was stained and analyzed by flow cytometry. % of the sub-G1 cell population is indicated as dead cells.

圖4A至4C係G2查核點廢除劑(l-Gly)(l-Arg)(l-Lys)(l-Lys)(l-Arg)(l-Arg)(l-Gln)(l-Arg)(l-Arg)(l-Cha)(l-Phe-2,3,4,5,6-F)(l-Arg)(l-Ser)(l-Pro)(l-Ser)(l-Tyr)(l-Tyr)(SEQ ID NO：78)之結構活性關係之示意圖：(A)在博來黴素處理之Jurkat細胞中針對l-Cha之胺基酸取代之G2查核點廢除活性按以下順序指示：[l-Cha=l-Nal(2)]>[l-Ala(3-Bzt)=l-Nal(1)=l-Trp=l-Dph]>[l-Ala(tBu)=Cys(tBu)=Leu]；(B)在秋水仙鹼處理之Jurkat細胞中針對l-Cha之胺基酸取代之M期查核點廢除活性及/或非特異性毒性之順序：[Ala(3-Bzt)=l-Nal(1)=l-Dph]>[l-Cha=l-Nal(2)]；(C)針對l-Phe-2,3,4,5,6-F之胺基酸取代之G2查核點廢除活性按以下順序指示：l-(Phe-2,3,4,5,6-F)=l-(Phe-3,4,5-F)=l-(Phe-4CF3)]>[l-(Phe-3Br,4Cl,5Br)=l-(Phe-4Cl)=l-Tyr]。 Figure 4A to 4C are G2 check point abolition agent (l-Gly) (l-Arg) (l-Lys) (l-Lys) (l-Arg) (l-Arg) (l-Gln) (l-Arg) (l-Arg)(l-Cha)(l-Phe-2,3,4,5,6-F)(l-Arg)(l-Ser)(l-Pro)(l-Ser)(l- Schematic representation of the structure-activity relationship of Tyr) (l-Tyr) (SEQ ID NO: 78): (A) G2 checkpoint abrogation activity by amino acid substitution for l-Cha in bleomycin-treated Jurkat cells The following order indicates: [l-Cha=l-Nal(2)]>[l-Ala(3-Bzt)=l-Nal(1)=l-Trp=l-Dph]>[l-Ala(tBu) =Cys(tBu)=Leu]; (B) Sequence of M-phase checkpoint abrogation of activity and/or non-specific toxicity for amino acid substitutions of 1-Cha in Jurkat cells treated with colchicine: [Ala( 3-Bzt)=l-Nal(1)=l-Dph]>[l-Cha=l-Nal(2)]; (C) against l-Phe-2,3,4,5,6-F The G2 checkpoint abrogation activity of amino acid substitutions is indicated in the following order: l-(Phe-2,3,4,5,6-F)=l-(Phe-3,4,5-F)=l-( Phe-4CF3)]>[l-(Phe-3Br,4Cl,5Br)=l-(Phe-4Cl)=l-Tyr].

圖5顯示各個富精胺酸序列之G2廢除活性。將所指示肽與或不與博來黴素一起添加至Jurkat細胞中。G2/M細胞%係在Y軸上指示。X軸係如下：1，單獨博來黴素；2，0.2μg/ml；3，0.39μg/ml；4，0.78μg/ml；5，1.56μg/ml；6，3.125μg/ml；7，6.25μg/ml；8，12.5μg/ml；9，25μg/ml；及10，50μg/ml。肽序列係如下：rrrqrrkkr， (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Lys)(d-Lys)(d-Arg)(SEQ ID NO：79)；CBP501，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：80)；no TAT，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：81)；rqrr，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：82)；rrqrr，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：83)；rrrq，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(SEQ ID NO：84)；及rrrqr，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(SEQ ID NO：85)。 Figure 5 shows the G2 abrogating activity of each arginine-rich sequence. The indicated peptides were added to Jurkat cells with or without bleomycin. The % G2/M cells are indicated on the Y-axis. The X axis is as follows: 1, bleomycin alone; 2, 0.2 μg/ml; 3, 0.39 μg/ml; 4, 0.78 μg/ml; 5, 1.56 μg/ml; 6, 3.125 μg/ml; 7, 6.25 μg/ml; 8, 12.5 μg/ml; 9, 25 μg/ml; and 10, 50 μg/ml. The peptide sequence is as follows: rrrqrrkkr, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)( d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Lys)(d-Lys)(d-Arg) (SEQ ID NO: 79 ); CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg )(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO:80); no TAT, (d-Bpa)(d-Ser)(d- Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO:81); rqrr,(d-Bpa)(d-Ser)( d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Gln)(d-Arg)(d-Arg )(SEQ ID NO: 82); rrqrr, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d -Cha)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO:83); rrrq,(d-Bpa)(d-Ser)(d -Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln) (SEQ ID NO: 84); and rrrqr, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d -Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg) (SEQ ID NO: 85).

圖6顯示各種無(d-Bpa)之肽之G2廢除活性。將所指示肽與或不與博來黴素一起添加至Jurkat細胞中。G2/M細胞%係在Y軸上指示。X軸係如下：1，單獨博來黴素；2，0.2μg/ml；3，0.39μg/ml；4，0.78μg/ml；5，1.56μg/ml；6，3.125μg/ml；7，6.25μg/ml；8，12.5μg/ml；9，25μg/ml；及10，50μg/ml。肽序列係如下：CBP0，(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：86)；CBP451，(d-Tyr)(d-Ser)(d-Pro)(l-Trp)(l-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：87)；CBP452，(d-Tyr)(d-Ser)(l-Pro)(l-Trp)(l-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：88)；及CBP501，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：80)。 Figure 6 shows the G2 abrogating activity of various peptides without (d-Bpa). The indicated peptides were added to Jurkat cells with or without bleomycin. The % G2/M cells are indicated on the Y-axis. The X axis is as follows: 1, bleomycin alone; 2, 0.2 μg/ml; 3, 0.39 μg/ml; 4, 0.78 μg/ml; 5, 1.56 μg/ml; 6, 3.125 μg/ml; 7, 6.25 μg/ml; 8, 12.5 μg/ml; 9, 25 μg/ml; and 10, 50 μg/ml. The peptide sequences are as follows: CBPO, (d-Arg) (d-Arg) (d-Arg) (d-Gln) (d-Arg) (d-Arg) (SEQ ID NO: 86); CBP451, (d- Tyr)(d-Ser)(d-Pro)(l-Trp)(l-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)( d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO:87); CBP452, (d-Tyr)(d-Ser)(l-Pro)( l-Trp)(l-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln )(d-Arg)(d-Arg)(SEQ ID NO:88); and CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3 ,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 80 ).

圖7顯示各個富精胺酸及富離胺酸之肽序列之G2廢除活性。將所指示肽添加至上述Jurkat細胞中並計算G2/M細胞%(Y軸)。肽序列係如下：CBP603，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe4NO2)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：89)；CBP607，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO：90)；CBP608，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO91：)；及CBP609，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Lys)(d-Lys)(d-Lys)(d-Lys)(d-Lys)(d-Lys)(SEQ ID NO：92)。 Figure 7 shows the G2 abrogating activity of various arginine-rich and lysine-rich peptide sequences. The indicated peptides were added to the above mentioned Jurkat cells and % G2/M cells were calculated (Y-axis). The peptide sequence is as follows: CBP603, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe4NO2)(d-Cha)(d-Arg)(d-Arg)(d- Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO:89); CBP607, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d- Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg) (SEQ ID NO: 90 ); CBP608, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg )(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO91: ); and CBP609, (d-Bpa)(d-Ser)(d-Trp )(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Lys)(d-Lys)(d-Lys)(d-Lys)(d -Lys)(d-Lys) (SEQ ID NO: 92).

圖8顯示序列之富精胺酸部分之位置可改變。將所指示肽添加至上述Jurkat細胞中且計算G2/M細胞%(Y軸)。肽序列係如下：CBP501，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：80)；CBP510，(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO：93)；CBP511，(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：94)；及CBP512，(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg) (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO：95)。 Figure 8 shows that the position of the arginine-rich portion of the sequence can be varied. The indicated peptides were added to the Jurkat cells above and % G2/M cells were calculated (Y-axis). The peptide sequence is as follows: CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)( d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO:80); CBP510, (d-Arg)(d-Arg)( d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp )(d-Ser)(d-Bpa)(SEQ ID NO:93); CBP511, (d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg )(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:94) and CBP512, (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg) (d-Cha)(d-Phe-2,3,4, 5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa) (SEQ ID NO: 95).

圖9顯示若干經研究經取代肽序列之結構。G2廢除活性在陰影較淺之取代下增加(*)，M期查核點廢除活性及/或非特異性毒性在陰影較深之取代下增加(**)且與其餘取代保持大約相同。 Figure 9 shows the structures of several investigated substituted peptide sequences. G2 abrogating activity increased with lighter shaded substitutions (*), M phase checkpoint abrogating activity and/or non-specific toxicity increased with darker shaded substitutions (**) and remained about the same as the remaining substitutions.

圖10顯示在Scid小鼠中利用CBP501及順鉑治療之後對腫瘤生長(人類胰臟癌)之抑制。第0天指示治療開始。對於每一治療組具有標準偏差之平均腫瘤大小在Y軸上指示且治療開始後之天數在X軸上指示。 Figure 10 shows inhibition of tumor growth (human pancreatic carcinoma) following treatment with CBP501 and cisplatin in Scid mice. Day 0 indicated the start of treatment. The mean tumor size with standard deviation for each treatment group is indicated on the Y-axis and the number of days after the start of treatment is indicated on the X-axis.

圖11顯示上述具有激酶抑制序列區及基於HIV-TAT轉導序列之序列區之肽之G2廢除活性。G2/M細胞%係在Y軸上指示。X軸係如下：1，單獨博來黴素；2，0.2μg/ml；3，0.39μg/ml；4，0.78μg/ml；5，1.56μg/ml；6，3.125μg/ml；7，6.25μg/ml；8，12.5μg/ml；9,25μg/ml；及10，50μg/ml。肽序列係如下：CBP501，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：80)；CBP700，(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：96)；CBP701，(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：97)；CBP702，(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：98)；CBP703，(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：99)。 Figure 11 shows the G2 abrogating activity of the above-mentioned peptides having the kinase inhibitory sequence region and the HIV-TAT transduction sequence-based sequence region. The % G2/M cells are indicated on the Y-axis. The X axis is as follows: 1, bleomycin alone; 2, 0.2 μg/ml; 3, 0.39 μg/ml; 4, 0.78 μg/ml; 5, 1.56 μg/ml; 6, 3.125 μg/ml; 7, 6.25 μg/ml; 8, 12.5 μg/ml; 9, 25 μg/ml; and 10, 50 μg/ml. The peptide sequence is as follows: CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)( d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 80); CBP700, (d-Arg)(d-Arg)( d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 96); CBP701 , (d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6 -F)(d-Cha)(SEQ ID NO:97); CBP702, (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d -Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 98); CBP703, (d-Arg)(d-Arg) (d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO: 99).

圖12顯示在肽之G2廢除活性與M廢除活性及/或非特異性毒性之間之比較，其中博來黴素用於G2廢除分析且秋水仙鹼用於M廢除活性及/或非特異性毒性。將所指示肽與博來黴素或秋水仙鹼一起添加至Jurkat細胞中。G2/M細胞%係在Y軸上指示。X軸係如下：1，單獨博來黴素或秋水仙鹼；2，0.2μg/ml；3，0.39μg/ml；4，0.78μg/ml；5，1.56μg/ml；6，3.125μg/ml；7，6.25μg/ml；8，12.5μg/ml；9，25μg/ml；及10，50μg/ml。肽序列係如下：CBP501，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)。 Figure 12 shows the comparison between G2 abolishing activity and M abolishing activity and/or non-specific toxicity of peptides, wherein bleomycin was used for G2 abolishing assay and colchicine was used for M abolishing activity and/or non-specific toxicity toxicity. The indicated peptides were added to Jurkat cells together with bleomycin or colchicine. The % G2/M cells are indicated on the Y-axis. The X axis is as follows: 1, bleomycin or colchicine alone; 2, 0.2 μg/ml; 3, 0.39 μg/ml; 4, 0.78 μg/ml; 5, 1.56 μg/ml; 6, 3.125 μg/ml ml; 7, 6.25 μg/ml; 8, 12.5 μg/ml; 9, 25 μg/ml; and 10, 50 μg/ml. The peptide sequence is as follows: CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)( d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg).

圖13顯示CBP501((d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg))之分子結構。 Figure 13 shows that CBP501 ((d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d- Molecular structure of Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)).

圖14顯示在所有經治療患者中關於基線WBC之整體存活之卡普蘭-邁耶分析(Kaplan-Meyer analysis)：在所有經治療患者中關於基線WBC之卡普蘭-邁耶存活曲線、中值OS及危險比。危險比隨截止含量降低而改良，且在WBC 8000/μl下之峰值作為截止含量。 Figure 14 shows Kaplan-Meyer analysis of overall survival on baseline WBC in all treated patients: Kaplan-Meyer survival curve on baseline WBC, median OS in all treated patients and hazard ratio. Hazard ratios improved with decreasing cut-off levels, and peaks at WBC 8000/μl were used as cut-off levels.

圖15顯示在ICON招募之患者中關於基線WBC之整體存活之卡普蘭-邁耶分析：在ICON招募患者中關於基線WBC之卡普蘭-邁耶存活曲線、中值OS及危險比。危險比隨截止含量降低而改良，且WBC 8000/μl下之峰值作為截止含量，且在A組與B組之間之差異在峰值下係統計顯著的。 Figure 15 shows Kaplan-Meier analysis of overall survival on baseline WBC in ICON-enrolled patients: Kaplan-Meier survival curves, median OS and hazard ratio on baseline WBC in ICON-enrolled patients. Hazard ratios improved with decreasing cut-off levels, and the peak at WBC 8000/μl was taken as the cut-off level, and the difference between groups A and B was statistically significant at the peak.

圖16顯示在各組顯示之所有經治療群體中篩選時，關於WBC(>8000或<8000)之卡普蘭-邁耶存活曲線、中值OS、患者數量、危險比及對數秩(Mantel-Cox)測試之p值。 Figure 16 shows Kaplan-Meier survival curves, median OS, number of patients, hazard ratio, and log-rank (Mantel-Cox) for WBC (>8000 or <8000) when screened in all treated populations shown in each group. ) test p-value.

圖17顯示藉由利用CBP501治療活化嗜中性球而增加之NET形成。 Figure 17 shows increased NET formation by treatment of activated neutrophils with CBP501.

圖18顯示在活體內藉由CBP501而增加之凝血酶/抗凝血酶複合物。 Figure 18 shows the increase of thrombin/antithrombin complex by CBP501 in vivo.

圖19A-19B顯示在活體外CBP501抑制之M1及M2巨噬細胞之吞噬作用。 Figures 19A-19B show CBP501 inhibited phagocytosis of M1 and M2 macrophages in vitro.

圖20顯示來自小鼠巨噬細胞系(RAW264.7)之TNF釋放之抑制。 Figure 20 shows inhibition of TNF release from a mouse macrophage cell line (RAW264.7).

圖21A-21C顯示CBP501可增強CT26 WT細胞中CDDP對細胞週期期分佈之效應。利用多個劑量之CDDP(X軸，ug/ml)與多個劑量之CBP501(0.0625μM、0.125μM、0.25μM、0.5μM及1μM)之組合將CT26WT細胞(鼠類結腸直腸細胞系)處理3小時，隨後用PBS洗滌並更換培養基。兩天後，藉由BD FACSCalibur藉由碘化丙啶染色來分析細胞週期期分佈(Y軸)及細胞死亡。 Figures 21A-21C show that CBP501 enhances the effect of CDDP on cell cycle phase distribution in CT26 WT cells. CT26WT cells (murine colorectal cell line) were treated with multiple doses of CDDP (X-axis, ug/ml) in combination with multiple doses of CBP501 (0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM and 1 μM)3 hours, followed by washing with PBS and changing the medium. Two days later, cell cycle phase distribution (Y axis) and cell death were analyzed by propidium iodide staining by BD FACSCalibur.

圖22A及22B顯示CBP501可增強CDDP誘導之eIF2-α磷酸化。利用10-20μM CDDP與0.5μM CBP501之組合將CT26WT細胞處理45min，隨後用PBS洗滌並添加新鮮培養基。一天後，收集細胞並藉由免疫印漬使用抗磷酸化eIF2-α之特定抗體進行分析(n=8)(圖2A)並對相對磷酸化eIF2-α之量進行定量(圖2B)。藉由司徒登氏t測試(student’s t-test)比較試樣。與未經處理之細胞相比較，*P<0.05，**P<0.01，且與單獨的相應CDDP相比較，^†P<0.05，^††P<0.01。 Figures 22A and 22B show that CBP501 enhances CDDP-induced eIF2-α phosphorylation. CT26WT cells were treated with a combination of 10-20 μM CDDP and 0.5 μM CBP501 for 45 min, followed by washing with PBS and addition of fresh medium. One day later, cells were harvested and analyzed by immunoblotting using a specific antibody against phosphorylated eIF2-α (n=8) ( FIG. 2A ) and the relative amount of phosphorylated eIF2-α was quantified ( FIG. 2B ). Samples were compared by student's t-test. *P<0.05, **P<0.01 compared to untreated cells, and ^† P<0.05, ^†† P<0.01 compared to the corresponding CDDP alone.

圖23顯示CBP501可增加CDDP誘導之細胞表面鈣網蛋白暴露。利用10-20μM CDDP與0.5μM CBP501之組合將CT26WT細胞處理45min，隨後用PBS洗滌並添加新鮮培養基。2天後，收集細胞並藉由FACS利用特定鈣網蛋白抗體及碘化丙啶進行分析(n=3)。藉由司徒登氏t測試比較試樣。與未經處理之細胞相比較，*P<0.05，**P<0.01，且與單獨的相應CDDP相比較，^†P<0.05，^††P<0.01。 Figure 23 shows that CBP501 increases CDDP-induced cell surface calreticulin exposure. CT26WT cells were treated with a combination of 10-20 μM CDDP and 0.5 μM CBP501 for 45 min, followed by washing with PBS and addition of fresh medium. After 2 days, cells were harvested and analyzed by FACS using specific calreticulin antibodies and propidium iodide (n=3). Samples were compared by Studen's t-test. *P<0.05, **P<0.01 compared to untreated cells, and ^† P<0.05, ^†† P<0.01 compared to the corresponding CDDP alone.

圖24顯示CBP501可加強CDDP誘導之HMGB1分泌。利用20μM CDDP與0.5μM CBP501之組合將CT26WT細胞處理45min，隨後用PBS洗滌並添加新鮮培養基。在處理後24小時、48小時及72小時收集分泌至培養基中之HMGB1(n=3)。藉由HMGB1 ELISA套組II執行經分泌HMGB1之定量。藉由司徒登氏t測試比較試樣。與未經處理之對照細胞相比較，*P<0.05，**P<0.01，且與單獨的相應CDDP相比較，^†P<0.05，^††P<0.01。 Figure 24 shows that CBP501 potentiates CDDP-induced HMGB1 secretion. CT26WT cells were treated with a combination of 20 μM CDDP and 0.5 μM CBP501 for 45 min, followed by washing with PBS and addition of fresh medium. HMGB1 secreted into the medium was collected at 24 hours, 48 hours and 72 hours after treatment (n=3). Quantification of secreted HMGB1 was performed by HMGB1 ELISA kit II. Samples were compared by Studen's t-test. *P<0.05, **P<0.01 compared to untreated control cells, and ^† P<0.05, ^†† P<0.01 compared to the corresponding CDDP alone.

圖25顯示CBP501可增強CDDP之抗腫瘤活性，且CDDP加CBP501可加強抗PD-1抗體之抗腫瘤活性。利用CBP501、CDDP及抗PD1抗體治療CT26WT s.c.同基因小鼠腫瘤模型。繪製腫瘤相對大小對開始治療後之天數之圖形。在第1天腫瘤之中值大小為0.1cm³(n=6)。箭頭表示治療天數(藍色箭頭/對於7.5mg/kg CBP501第1天、第2天、第8天、第9天、第15天及第16天，紅色箭頭/對於5mg/kg CDDP第2天、第9天及第16天及粉色箭頭/對於200ug抗PD1第3天、第10天及第17天)。在第23天在第2組中觀察到1隻小鼠死亡，在第26天在第5組中觀察到1隻小鼠死亡，且在第28天在第5組中觀察到1隻小鼠死亡。 Figure 25 shows that CBP501 can enhance the anti-tumor activity of CDDP, and CDDP plus CBP501 can enhance the anti-tumor activity of anti-PD-1 antibody. CT26WT sc syngeneic mouse tumor model was treated with CBP501, CDDP and anti-PD1 antibody. The relative tumor size is plotted versus the number of days since the start of treatment. The median tumor size was 0.1 cm ³ (n=6) on day 1. Arrows indicate days of treatment (blue arrows/days 1, 2, 8, 9, 15, and 16 for 7.5 mg/kg CBP501, red arrows/day 2 for 5 mg/kg CDDP , day 9 and day 16 and pink arrows / day 3, day 10 and day 17 for 200ug anti-PD1). 1 mouse death was observed in group 2 on day 23, 1 mouse death was observed in group 5 on day 26, and 1 mouse was observed in group 5 on day 28 die.

圖26顯示在異體同質異種移植物模型小鼠中腫瘤生長受到CBP501、順鉑及抗PD1抗體中之每一者及CBP501、順鉑及抗PD1抗體之組合之抑制。 Figure 26 shows that tumor growth was inhibited by each of CBP501, cisplatin and anti-PD1 antibody and the combination of CBP501, cisplatin and anti-PD1 antibody in allogeneic xenograft model mice.

圖27顯示異種移植物研究中之體重變化及個別腫瘤之生長。 Figure 27 shows changes in body weight and growth of individual tumors in a xenograft study.

圖28顯示在異體同質異種移植物小鼠模型中腫瘤生長受到CBP501、順鉑及抗PD-L1抗體中之每一者及其組合之抑制。 Figure 28 shows that tumor growth was inhibited by each of CBP501, cisplatin, and anti-PD-L1 antibody, and combinations thereof, in an allogeneic xenograft mouse model.

圖29顯示異種移植物研究中之體重變化及個別腫瘤之生長。 Figure 29 shows changes in body weight and growth of individual tumors in a xenograft study.

圖30顯示在異體同質異種移植物小鼠模型中腫瘤生長受到CBP501、卡鉑及抗PD1抗體中之每一者及其組合之抑制。 Figure 30 shows that tumor growth was inhibited by each of CBP501, carboplatin, and anti-PD1 antibody, and combinations thereof, in an allogeneic xenograft mouse model.

圖31顯示異種移植物研究中之體重變化及個別腫瘤之生長。 Figure 31 shows changes in body weight and growth of individual tumors in a xenograft study.

圖32顯示對腫瘤浸潤性淋巴球(TIL)之分析。 Figure 32 shows the analysis of tumor infiltrating lymphocytes (TIL).

相關申請案資訊Related Application Information

此申請案主張在2015年10月23日提出申請之美國臨時專利申請案第62/245,899號及在2016年6月3日提出申請之美國臨時專利申請案第62/345,416號之優先權。上述申請案之全部內容(包括所有文本、表格及附圖)係以引用方式併入本文中。 This application claims priority to U.S. Provisional Patent Application No. 62/245,899, filed October 23, 2015, and U.S. Provisional Patent Application No. 62/345,416, filed June 3, 2016. The entire contents of the above application, including all text, tables and figures, are hereby incorporated by reference.

本發明提供化合物之組合，及單獨化合物及化合物之各種組合之方法及用途。一種該組合、方法或用途包括如本文所闡述之T細胞活化劑及肽或擬肽。另一該組合、方法或用途包括如本文所闡述之免疫查核點抑制劑及肽或擬肽。又一該組合、方法或用途包括如本文所闡述之T細胞活化劑及免疫查核點抑制劑。再一該組合、方法或用途包括如本文所闡述之T細胞活化劑及免疫查核點抑制劑及肽或擬肽。因此，本發明化合物之組合、及化合物單獨及於各種組合中之方法及用途可用於治療特徵在於不合意或不期望細胞增殖(例如良性及惡性腫瘤細胞)之細胞增殖病症或生理病況。 The present invention provides combinations of compounds, and methods and uses of individual compounds and various combinations of compounds. One such combination, method or use comprises a T cell activator and a peptide or peptidomimetic as described herein. Another such combination, method or use comprises an immune checkpoint inhibitor and a peptide or peptidomimetic as described herein. Yet another such combination, method or use comprises a T cell activator and an immune checkpoint inhibitor as described herein. Yet another such combination, method or use includes a T cell activator and an immune checkpoint inhibitor and a peptide or peptidomimetic as described herein. Combinations of compounds of the invention, and methods and uses of the compounds alone and in various combinations, are thus useful in the treatment of cell proliferative disorders or physiological conditions characterized by undesirable or undesired proliferation of cells, such as benign and malignant tumor cells.

組合包括抑制細胞增殖之肽及擬肽。肽及擬肽抑制細胞增殖之能力似乎至少部分地由廢除細胞週期G2查核點引起。由於細胞可藉由抑制G2查核點因應核酸損害而被誘導進入細胞週期G2查核點以容許細胞在發生DNA複製及細胞分裂前修復損害，故本發明肽及擬肽可使細胞對核酸損害劑及治療方案敏感。累積足夠核酸損害之細胞將由於G2查核點中斷而無法完全修復受損核酸。該等細胞將展現降低之增殖(例如，由未修復之對於存活關鍵之基因之突變引起)且最後經歷細胞凋亡。 Combinations include peptides and peptidomimetics that inhibit cell proliferation. The ability of peptides and peptidomimetics to inhibit cell proliferation appears to result, at least in part, from the abrogation of the cell cycle G2 checkpoint. Since cells can be induced to enter the G2 checkpoint of the cell cycle in response to nucleic acid damage by inhibiting the G2 checkpoint to allow cells to repair the damage before DNA replication and cell division occur, the peptides and peptidomimetics of the present invention can make cells resistant to nucleic acid damage agents and Treatment options are sensitive. Cells that have accumulated sufficient nucleic acid damage will be unable to fully repair damaged nucleic acid due to disruption of the G2 checkpoint. These cells will exhibit reduced proliferation (eg, caused by unrepaired mutations in genes critical for survival) and eventually undergo apoptosis.

具有正常G1之細胞較不易於累積受損核酸，乃因核酸修復亦可在G1期間進行。因此，正常細胞對本發明化合物之影響較不敏感。然而，具有受損或經中斷細胞週期G1查核點之細胞較可能累積受損核酸，乃因G1查核點受損或中斷使得該等細胞較不可能完全修復受損核酸。因此，利用中斷G2查核點之本發明肽或擬肽處理G1受損或中斷之細胞使得該等細胞甚至更不可能完全修復受損核酸。因此，G1受損或中斷之細胞對該等本發明肽及擬肽特別敏感。因此，本發明化合物(包括肽及擬肽)一般可用於抑制或預防細胞增殖且具體而言抑制具有受損或中斷之G1查核點之細胞之增殖。 Cells with normal G1 are less prone to accumulating damaged nucleic acids because nucleic acid repair can also occur during G1. Therefore, normal cells are less sensitive to the effects of the compounds of the invention. However, cells with damaged or disrupted cell cycle G1 checkpoints are more likely to accumulate damaged nucleic acids because damaged or disrupted G1 checkpoints make it less likely for these cells to fully repair damaged nucleic acids. Thus, treatment of G1-impaired or disrupted cells with peptides or peptidomimetics of the invention that disrupt the G2 checkpoint makes these cells even less likely to fully repair damaged nucleic acids. Thus, cells with impaired or disrupted G1 are particularly sensitive to these peptides and peptidomimetics of the invention. Accordingly, compounds of the invention, including peptides and peptidomimetics, are useful for inhibiting or preventing cell proliferation in general and, in particular, inhibiting the proliferation of cells with an impaired or disrupted Gl checkpoint.

具有受損或中斷之G1細胞週期查核點之細胞包括(但不限於)快速增殖之細胞。特徵在於細胞生長較快、細胞生長不合意或細胞存活而非經歷細胞凋亡之細胞增殖病症及生理病況通常具有受損或中斷之G1細胞週期查核點。因此，在本發明肽及擬肽抑制增殖或刺激細胞凋亡之能力似乎至少部分地由中斷G2細胞週期查核點引起時，因G1查核點受損或中斷而快速或不合意增殖之細胞係特別有吸引力的靶標。 Cells with an impaired or disrupted G1 cell cycle checkpoint include, but are not limited to, rapidly proliferating cells. Cell proliferative disorders and physiological conditions characterized by faster cell growth, undesirable cell growth, or cell survival rather than undergoing apoptosis often have an impaired or disrupted G1 cell cycle checkpoint. Thus, while the ability of the peptides and peptidomimetics of the invention to inhibit proliferation or stimulate apoptosis appears to be caused, at least in part, by disruption of the G2 cell cycle checkpoint, cell lines that proliferate rapidly or undesirable due to impaired or disrupted G1 checkpoint are particularly attractive target.

CBP501係細胞週期G2查核點抑制性肽TAT-S216A(Suganuma,M.等人Cancer Res.59：5887(1999))。採用基於細胞週期表型之篩選方法來最佳化TAT-S216A以因應DNA損害劑而在細胞週期G2期減少癌細胞之累積，而不影響正常細胞之細胞週期表型(Sha,S.等人Mol.Cancer Ther.6：147(2007))。發現CBP501可使CBP501敏感性腫瘤細胞中鉑濃度及鉑-DNA加成物形成增加，且或者或除G2查核點抑制/中斷以外可經由攜製鈣蛋白抑制起作用(Mine,N.等人Mol.Cancer Ther.10：1929(2011))。 CBP501 is a cell cycle G2 checkpoint inhibitory peptide TAT-S216A (Suganuma, M. et al. Cancer Res. 59:5887 (1999)). A cell cycle phenotype-based screening approach was used to optimize TAT-S216A to reduce the accumulation of cancer cells in the G2 phase of the cell cycle in response to DNA damaging agents without affecting the cell cycle phenotype of normal cells (Sha, S. et al. Mol. Cancer Ther. 6:147 (2007)). CBP501 was found to increase platinum concentration and platinum-DNA adduct formation in CBP501-sensitive tumor cells, and either or in addition to G2 checkpoint inhibition/disruption could act via calpain inhibition (Mine, N. et al. Mol . Cancer Ther. 10:1929 (2011)).

本發明化合物(包括肽及擬肽)本身可在不利用損害核酸或具有抗增殖活性之其他治療下抑制細胞增殖，乃因在細胞分裂時中斷G2查核點將可能引起核酸損害累積。因此，可利用單獨的本發明化合物或與核酸損害治療(例如，化學藥劑或治療方案)之組合處理異常或不合意增殖或存活之細胞，以抑制或預防細胞增殖或刺激細胞凋亡/劇變。 The compounds of the present invention, including peptides and peptidomimetics, can inhibit cell proliferation by themselves without the use of other treatments that damage nucleic acids or have antiproliferative activity, because disruption of the G2 checkpoint during cell division would make it possible Causes accumulation of nucleic acid damage. Accordingly, abnormal or undesirable proliferating or surviving cells can be treated with compounds of the invention alone or in combination with nucleic acid damaging treatments (eg, chemical agents or therapeutic regimens) to inhibit or prevent cell proliferation or stimulate apoptosis/catastrophe.

與無論細胞係正常的抑或異常的(例如，癌細胞)均快速靶向增殖細胞之習用抗細胞增殖劑不同，本發明化合物優先靶向具有受損或中斷之細胞週期G1查核點之細胞。舉例而言，CBP501不影響HUVEC細胞之生長(例如，參見表3)。CBP501亦不影響由秋水仙鹼誘導之M期細胞週期停滯及/或非特異性毒性(例如，參見圖12)。因此，本發明化合物較不可能產生過量與習用抗細胞增殖治療劑相關之不合意副作用，例如骨髓抑制、噁心、食欲缺乏、腹瀉及掉發。另外，由於絕大多數癌細胞具有受損或中斷之細胞週期G1查核點，故癌細胞對廢除細胞週期G2查核點之本發明化合物之敏感性將增加。那麼正常細胞較不受影響亦意味著包括肽及擬肽之本發明化合物可以較大量使用。 Unlike conventional anti-cell proliferation agents that rapidly target proliferating cells, whether the cell line is normal or abnormal (eg, cancer cells), the compounds of the invention preferentially target cells with impaired or disrupted cell cycle G1 checkpoint. For example, CBP501 did not affect the growth of HUVEC cells (eg, see Table 3). CBP501 also did not affect M-phase cell cycle arrest and/or non-specific toxicity induced by colchicine (eg, see Figure 12). Thus, the compounds of the present invention are less likely to produce undesired side effects, such as myelosuppression, nausea, anorexia, diarrhea and hair loss, associated with excess of conventional anti-cell proliferation therapeutics. In addition, since the vast majority of cancer cells have an impaired or disrupted cell cycle G1 checkpoint, the sensitivity of cancer cells to compounds of the invention that abolish the cell cycle G2 checkpoint will be increased. The fact that normal cells are less affected also means that the compounds of the invention, including peptides and peptidomimetics, can be used in larger quantities.

根據本發明，提供具有抗細胞增殖活性及/或廢除G2細胞週期查核點之化合物(包括肽及擬肽)。肽或擬肽包括抑制細胞之增殖或刺激細胞之細胞凋亡之序列。肽或擬肽亦包括廢除細胞週期G2查核點之序列。在一個實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、P4、P5、P6(SEQ ID NO：1)或P6、P5、P4、P3、P2、P1(SEQ ID NO：2)；其中P1係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、佔據類似側鏈空間之胺基酸(例如，d-Tyr或l-Tyr、d-Phe或l-Phe)，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘基團、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉、喹唑啉基團之任一胺基酸；P2係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、佔據類似側鏈空間之胺基酸(例如d-Tyr或l-Tyr、d-Phe或l-Phe)，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

烷、喹喏啉或喹唑啉基團之任一胺基酸；P3、P4、P5係任一胺基酸或P3、P4、P5中之一或多者係簡單碳鏈，使得P2與P6之間之距離與當P3、P4、P5中之每一者係胺基酸時之距離大約相同(d-Trp或l-Trp係P4處之實例；P6係d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、任一胺基酸及d-Tyr或l-Tyr(例如，d-Ser-d-Tyr)、任一胺基酸及d-Phe或l-Phe(例如，d-Ser-d-Phe)、任一胺基酸，或不存在。在各個態樣中，具有簡單碳鏈之胺基酸係d-11-胺基十一酸或l-11-胺基十一酸、d-10-胺基癸酸或l-10-胺基癸酸、d-9-胺基壬酸或l-9-胺基壬酸、d-8-胺基辛酸或l-8-胺基辛酸、d-7-胺基庚酸或l-7-胺基庚酸、d-6-胺基己酸或l-6-胺基己酸或具有一或多個不飽和碳鍵之類似結構。 According to the present invention, compounds (including peptides and peptidomimetics) having anti-cell proliferative activity and/or abrogating the G2 cell cycle checkpoint are provided. Peptides or peptidomimetics include sequences that inhibit proliferation of cells or stimulate apoptosis of cells. Peptides or peptidomimetics also include sequences that abolish the G2 checkpoint of the cell cycle. In one embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6 (SEQ ID NO: 1) or P6, P5, P4, P3, P2, P1 (SEQ ID NO : 2); wherein P1 is d-Cha or l-Cha, d-Nal(2) or l-Nal(2), d-(Phe-2,3,4,5,6-F) or l-( Phe-2,3,4,5,6-F), d-(Phe-3,4,5F) or l-(Phe-3,4,5F), d-(Phe-4CF3) or l-( Phe-4CF3), amino acids occupying similar side chain spaces (e.g., d-Tyr or l-Tyr, d-Phe or l-Phe), or having one or two aromatic, hexahydropyridine in the side chain , pyrazine, pyrimidine, hexahydropyrazine, morpholine or pyrimidine group or an indole, pentalene, indene, naphthalene group, benzofuran, benzothiophene, quinoline, indoline,

Any amino acid of 锍, quinoxaline, quinazoline group; P2 is d-Cha or l-Cha, d-Nal(2) or l-Nal(2), d-(Phe-2,3 ,4,5,6-F) or l-(Phe-2,3,4,5,6-F), d-(Phe-3,4,5F) or l-(Phe-3,4,5F ), d-(Phe-4CF3) or l-(Phe-4CF3), d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, amino acids occupying similar side chain spaces (such as d-Tyr or l -Tyr, d-Phe or l-Phe), or have one or two aromatic, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or pyrimidine groups in the side chain or an indole, Pentalene, indene, naphthalene, benzofuran, benzothiophene, quinoline, indoline,

Any amino acid of alkane, quinoxaline or quinazoline group; P3, P4, P5 is any amino acid or one or more of P3, P4, P5 is a simple carbon chain, so that P2 and P6 The distance between them is about the same as when each of P3, P4, P5 is an amino acid (d-Trp or l-Trp is an example at P4; P6 is d-Bpa or l-Bpa, d -Phe or l-Phe4NO2, any amino acid and d-Tyr or l-Tyr (for example, d-Ser-d-Tyr), any amino acid and d-Phe or l-Phe (for example, d- Ser-d-Phe), any amino acid, or not present. In each case, the amino acid with a simple carbon chain is d-11-aminoundecanoic acid or 1-11-aminoundecanoic acid acid, d-10-aminodecanoic acid or l-10-aminodecanoic acid, d-9-aminononanoic acid or l-9-aminononanoic acid, d-8-aminocaprylic acid or l-8- Aminocaprylic acid, d-7-aminoheptanoic acid or l-7-aminoheptanoic acid, d-6-aminocaproic acid or l-6-aminocaproic acid or those having one or more unsaturated carbon bonds similar structure.

在另一實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、P4、P5、P6(SEQ ID NO：3)；P6、P5、P4、P3、P2、P1(SEQ ID NO：4)；P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12(SEQ ID NO：5)；P1、P2、P3、P4、P5、P6、P12、P11、P10、P9、P8、P7(SEQ ID NO：6)；P6、P5、P4、P3、P2、P1、P7、P8、P9、P10、P11、P12(SEQ ID NO：7)；P6、P5、P4、P3、P2、P1、P12、P11、P10、P9、P8、P7(SEQ ID NO：8)；P7、P8、P9、P10、P11、P12、P1、P2、P3、P4、P5、P6(SEQ ID NO：9)； P7、P8、P9、P10、P11、P12、P6、P5、P4、P3、P2、P1(SEQ ID NO：10)；P12、P11、P10、P9、P8、P7、P1、P2、P3、P4、P5、P6(SEQ ID NO：11)；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1(SEQ ID NO：12)；P12、P11、P6、P9、P8、P7、P2、P1(SEQ ID NO：13)；P12、P11、P10、P6、P9、P4、P7、P2、P1(SEQ ID NO：14)；P1、P2、P7、P8、P9、P6、P11、P12(SEQ ID NO：15)；或P1、P2、P7、P4、P9、P6、P10、P11、P12(SEQ ID NO：16)；其中P1係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、佔據類似側鏈空間之胺基酸(例如d-Tyr或l-Tyr、d-Phe或l-Phe)，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉或喹唑啉基團之任一胺基酸；P2係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)或佔據類似側鏈空間之胺基酸(例如d-Tyr或l-Tyr、d-Phe或l-Phe)或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團、或一個吲哚、并環戊二烯、茚、萘基團、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉、喹唑啉基團之任一胺基酸；P3、P4、P5係任一胺基酸或P3、P4、P5中之一或多者係簡單碳鏈，使得P2與P6之間之距離與當P3、P4、P5中之每一者係胺基酸時之距離大約相同(d-Trp或l-Trp係P4處之實例)；P6係d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、任一胺基酸及d-Tyr或l-Tyr(例如，d-Ser-d-Tyr)、任一胺基酸及d-Phe或 l-Phe(例如，d-Ser-d-Phe)，且P7、P8、P9、P10、P11、P12中之至少三者係鹼性胺基酸，且其餘為任一胺基酸或不存在。在各個態樣中，具有簡單碳鏈之胺基酸係d-11-胺基十一酸或l-11-胺基十一酸、d-10-胺基癸酸或l-10-胺基癸酸、d-9-胺基壬酸或l-9-胺基壬酸，d-8-胺基辛酸或l-8-胺基辛酸、d-7-胺基庚酸或l-7-胺基庚酸、d-6-胺基己酸或l-6-胺基己酸或具有一或多個不飽和碳鍵之類似結構。 In another embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6 (SEQ ID NO: 3); P6, P5, P4, P3, P2, P1 (SEQ ID NO: 3); NO: 4); P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12 (SEQ ID NO: 5); P1, P2, P3, P4, P5, P6, P12, P11, P10, P9, P8, P7 (SEQ ID NO: 6); P6, P5, P4, P3, P2, P1, P7, P8, P9, P10, P11, P12 (SEQ ID NO: 7); P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8, P7 (SEQ ID NO: 8); P7, P8, P9, P10, P11, P12, P1, P2, P3, P4, P5 , P6 (SEQ ID NO: 9); P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 10); P12, P11, P10, P9, P8 , P7, P1, P2, P3, P4, P5, P6 (SEQ ID NO: 11); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 12); P12, P11, P6, P9, P8, P7, P2, P1 (SEQ ID NO: 13); P12, P11, P10, P6, P9, P4, P7, P2, P1 (SEQ ID NO: 14) ; P1, P2, P7, P8, P9, P6, P11, P12 (SEQ ID NO: 15); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO: 16); Among them, P1 is d-Cha or l-Cha, d-Nal(2) or l-Nal(2), d-(Phe-2,3,4,5,6-F) or l-(Phe-2, 3,4,5,6-F), d-(Phe-3,4,5F) or l-(Phe-3,4,5F), d-(Phe-4CF3) or l-(Phe-4CF3) , d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, amino acids occupying similar side chain spaces (such as d-Tyr or l-Tyr, d-Phe or l-Phe), or in side chains With one or two aromatic, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or pyrimidine groups or an indole, pentalene, indene, naphthalene, benzofuran, benzothiophene , quinoline, indoline,

Any amino acid of N, quinoline or quinazoline group; P2 is d-Cha or l-Cha, d-Nal(2) or l-Nal(2), d-(Phe-2,3 ,4,5,6-F) or l-(Phe-2,3,4,5,6-F), d-(Phe-3,4,5F) or l-(Phe-3,4,5F ), d-(Phe-4CF3) or l-(Phe-4CF3) or an amino acid occupying a similar side chain space (such as d-Tyr or l-Tyr, d-Phe or l-Phe) or in the side chain having one or two aromatic, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or pyrimidine groups, or an indole, pentalene, indene, naphthalene, benzofuran, Benzothiophene, quinoline, indoline,

Any amino acid of N, quinoline, quinazoline group; P3, P4, P5 is any amino acid or one or more of P3, P4, P5 is a simple carbon chain, so that P2 and P6 The distance between them is about the same as when each of P3, P4, P5 is an amino acid (d-Trp or l-Trp is an example at P4); P6 is d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, any amino acid and d-Tyr or l-Tyr (for example, d-Ser-d-Tyr), any amino acid and d-Phe or l-Phe (for example, d -Ser-d-Phe), and at least three of P7, P8, P9, P10, P11, and P12 are basic amino acids, and the rest are any amino acids or do not exist. In various aspects, the amino acids with simple carbon chains are d-11-aminoundecanoic acid or l-11-aminoundecanoic acid, d-10-aminodecanoic acid or l-10-amino Capric acid, d-9-aminononanoic acid or l-9-aminononanoic acid, d-8-aminocaprylic acid or l-8-aminocaprylic acid, d-7-aminoheptanoic acid or l-7- Aminoheptanoic acid, d-6-aminocaproic acid or 1-6-aminocaproic acid or similar structures with one or more unsaturated carbon bonds.

在另一實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12(SEQ ID NO：17)；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1(SEQ ID NO：18)；P12、P11、P10、P6、P9、P4、P7、P2、P1(SEQ ID NO：19)；或P1、P2、P7、P4、P9、P6、P10、P11、P12(SEQ ID NO：20)；其中P1係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、佔據類似側鏈空間之胺基酸(例如d-Tyr或l-Tyr、d-Phe或l-Phe)，或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉或喹唑啉基團之任一胺基酸；P2係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、佔據類似側鏈空間之胺基酸(例如d-Tyr或l-Tyr、d-Phe或l-Phe)或在側鏈中具有一或兩個芳香族、六氫吡啶、吡嗪、嘧啶、六氫吡嗪、嗎啉或嘧啶基團或一個吲哚、并環戊二烯、茚、萘、苯并呋喃、苯并噻吩、喹啉、吲哚啉、

唍、喹喏啉、喹唑啉基團之任一胺基酸；P3、P4、 P5係任一胺基酸或P3、P4、P5中之一或多者係簡單碳鏈，使得P2與P6之間之距離與當P3、P4、P5中之每一者係胺基酸時之距離大約相同(d-Trp或l-Trp係P4處之實例)；P6係d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、任一胺基酸及d-Tyr或l-Tyr(例如，d-Ser-d-Tyr)、任一胺基酸及d-Phe或l-Phe(例如，d-Ser-d-Phe)、任一胺基酸，或不存在；且P7、P8、P9、P10、P11、P12中之至少三者係鹼性胺基酸，且其餘為任一胺基酸或不存在。在各個態樣中，具有簡單碳鏈之胺基酸係d-胺基十一酸或l-胺基十一酸或d-8-胺基辛酸或l-8-胺基辛酸。 In another embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12 (SEQ ID NO: 17); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 18); P12, P11, P10, P6, P9, P4, P7, P2, P1 (SEQ ID NO : 19); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO: 20); wherein P1 is d-Cha or l-Cha, d-Nal(2) or l- Nal(2), d-(Phe-2,3,4,5,6-F) or l-(Phe-2,3,4,5,6-F), d-(Phe-3,4, 5F) or l-(Phe-3,4,5F), d-(Phe-4CF3) or l-(Phe-4CF3), d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, occupying similar side Amino acids in the chain space (such as d-Tyr or l-Tyr, d-Phe or l-Phe), or with one or two aromatics, hexahydropyridine, pyrazine, pyrimidine, hexahydropyridine in the side chain an oxazine, morpholine or pyrimidine group or an indole, pentalene, indene, naphthalene, benzofuran, benzothiophene, quinoline, indoline,

Any amino acid of N, quinoline or quinazoline group; P2 is d-Cha or l-Cha, d-Nal(2) or l-Nal(2), d-(Phe-2,3 ,4,5,6-F) or l-(Phe-2,3,4,5,6-F), d-(Phe-3,4,5F) or l-(Phe-3,4,5F ), d-(Phe-4CF3) or l-(Phe-4CF3), amino acids occupying similar side chain spaces (such as d-Tyr or l-Tyr, d-Phe or l-Phe) or in side chains With one or two aromatic, hexahydropyridine, pyrazine, pyrimidine, hexahydropyrazine, morpholine or pyrimidine groups or an indole, pentalene, indene, naphthalene, benzofuran, benzothiophene , quinoline, indoline,

Any amino acid of N, quinoxaline, quinazoline group; P3, P4, P5 is any amino acid or one or more of P3, P4, P5 is a simple carbon chain, so that P2 and P6 The distance between them is about the same as when each of P3, P4, P5 is an amino acid (d-Trp or l-Trp is an example at P4); P6 is d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, any amino acid and d-Tyr or l-Tyr (for example, d-Ser-d-Tyr), any amino acid and d-Phe or l-Phe (for example, d -Ser-d-Phe), any amino acid, or not present; and at least three of P7, P8, P9, P10, P11, and P12 are basic amino acids, and the rest are any amino acids or does not exist. In various aspects, the amino acid with a simple carbon chain is d-aminoundecanoic acid or 1-aminoundecanoic acid or d-8-aminooctanoic acid or 1-8-aminooctanoic acid.

在另一實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、P4、P5、P6(SEQ ID NO：21)或P6、P5、P4、P3、P2、P1(SEQ ID NO：22)；其中P1係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、d-Tyr或l-Tyr或d-Phe或l-Phe；P2係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、d-Tyr或l-Tyr或d-Phe或l-Phe；P3係d-絲胺酸或l-絲胺酸、d-精胺酸或l-精胺酸、d-半胱胺酸或l-半胱胺酸、d-脯胺酸或l-脯胺酸或d-天冬醯胺或l-天冬醯胺；P4係d-色胺酸或l-色胺酸；且P5係d-絲胺酸或l-絲胺酸、d-精胺酸或l-精胺酸，或d-天冬醯胺或l-天冬醯胺；或P3、P4、P5係單一d-胺基十一酸或l-胺基十一酸或單一d-8-胺基辛酸或l-8-胺基辛酸；P6係d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、(d-Ser-d-Tyr)或(d-Ser-d-Phe)。 In another embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6 (SEQ ID NO: 21) or P6, P5, P4, P3, P2, P1 (SEQ ID NO: 22); where P1 is d-Cha or l-Cha, d-Nal(2) or l-Nal(2), d-(Phe-2,3,4,5,6-F) or l- (Phe-2,3,4,5,6-F), d-(Phe-3,4,5F) or l-(Phe-3,4,5F), d-(Phe-4CF3) or l- (Phe-4CF3), d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, d-Tyr or l-Tyr or d-Phe or l-Phe; P2 is d-Cha or l-Cha, d- Nal(2) or l-Nal(2), d-(Phe-2,3,4,5,6-F) or l-(Phe-2,3,4,5,6-F), d- (Phe-3,4,5F) or l-(Phe-3,4,5F), d-(Phe-4CF3) or l-(Phe-4CF3), d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, d-Tyr or l-Tyr or d-Phe or l-Phe; P3 is d-serine or l-serine, d-arginine or l-arginine, d-cysteine amino acid or l-cysteine, d-proline or l-proline or d-asparagine or l-asparagine; P4 is d-tryptophan or l-tryptophan; And P5 is d-serine or l-serine, d-arginine or l-arginine, or d-asparagine or l-asparagine; or P3, P4, P5 are single d-aminoundecanoic acid or l-aminoundecanoic acid or single d-8-aminooctanoic acid or l-8-aminooctanoic acid; P6 is d-Bpa or l-Bpa, d-Phe or l-Phe4NO2 , (d-Ser-d-Tyr) or (d-Ser-d-Phe).

在另一實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、 P4、P5、P6、P7、P8、P9、P10、P11、P12(SEQ ID NO：23)；P1、P2、P3、P4、P5、P6、P12、P11、P10、P9、P8、P7(SEQ ID NO：24)；P6、P5、P4、P3、P2、P1、P7、P8、P9、P10、P11、P12(SEQ ID NO：25)；P6、P5、P4、P3、P2、P1、P12、P11、P10、P9、P8、P7(SEQ ID NO：26)；P7、P8、P9、P10、P11、P12、P1、P2、P3、P4、P5、P6(SEQ ID NO：27)；P7、P8、P9、P10、P11、P12、P6、P5、P4、P3、P2、P1(SEQ ID NO：28)；P12、P11、P10、P9、P8、P7、P1、P2、P3、P4、P5、P6(SEQ ID NO：29)；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1(SEQ ID NO：30)；P12、P11、P6、P9、P8、P7、P2、P1(SEQ ID NO：31)；P12、P11、P10、P6、P9、P4、P7、P2、P1(SEQ ID NO：32)；P1、P2、P7、P8、P9、P6、P11、P12(SEQ ID NO：33)；或P1、P2、P7、P4、P9、P6、P10、P11、P12(SEQ ID NO：34)；其中P1係d-Cha或l-Cha、Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、d-Tyr或l-Tyr或d-Phe或l-Phe；P2係d-Cha或l-Cha、d-Nal(2)或l-Nal(2)、d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)、d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、d-Tyr或l-Tyr或d-Phe或l-Phe；P3係d-絲胺酸或l-絲胺酸、d-精胺酸或l-精胺酸、d-半胱胺酸或l-半胱胺酸、d-脯胺酸或l-脯胺酸，或d-天冬醯胺或l-天冬醯胺；P4係d-色胺酸或l-色胺酸；P5係d-絲胺酸或l-絲胺酸、d-精胺酸或l-精胺酸，或d-天冬醯胺或l-天冬醯胺；或P3、P4、P5係單一d-胺基十一酸或l-胺基十一酸或單一d-8-胺基辛酸或l-8-胺基辛酸；P6係d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、(d-Ser-d-Tyr)或(d-Ser-d-Phe)；且P7、P8、P9、P10、P11、 P12中之至少三者係d-Arg或l-Arg或d-Lys或l-Lys，且其餘為任一胺基酸或不存在。 In another embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12 (SEQ ID NO: 23); P1, P2, P3, P4, P5, P6, P12, P11, P10, P9, P8, P7 (SEQ ID NO: 23); ID NO: 24); P6, P5, P4, P3, P2, P1, P7, P8, P9, P10, P11, P12 (SEQ ID NO: 25); P6, P5, P4, P3, P2, P1, P12 , P11, P10, P9, P8, P7 (SEQ ID NO: 26); P7, P8, P9, P10, P11, P12, P1, P2, P3, P4, P5, P6 (SEQ ID NO: 27); P7 , P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 28); P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6 (SEQ ID NO: 29); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 30); P12, P11, P6, P9, P8, P7, P2, P1 (SEQ ID NO: 31); P12, P11, P10, P6, P9, P4, P7, P2, P1 (SEQ ID NO: 32); P1, P2, P7, P8, P9, P6, P11, P12 (SEQ ID NO: 33); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO: 34); wherein P1 is d-Cha or l-Cha, Nal(2), d-(Phe-2,3,4,5,6-F) or l-(Phe-2,3,4,5,6-F), d-(Phe-3,4, 5F) or l-(Phe-3,4,5F), d-(Phe-4CF3) or l-(Phe-4CF3), d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, d-Tyr Or l-Tyr or d-Phe or l-Phe; P2 is d-Cha or l-Cha, d-Nal(2) or l-Nal(2), d-(Phe-2,3,4,5, 6-F) or l-(Phe-2,3,4,5,6-F), d-(Phe-3,4,5F) or l-(Phe-3,4,5F), d-( Phe-4CF3) or l-(Phe-4CF3), d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, d-Tyr or l-Tyr or d-Phe or l-Phe; P3 is d-silk amino acid or l-serine, d-arginine or l-arginine, d-cysteine or l-cysteine, d-proline or l-proline, or d- Asparagine or l-asparagine; P4 is d-tryptophan or l-tryptophan; P5 is d-serine or l-serine, d-arginine or l-spermine Acid, or d-asparagine or l-asparagine; or P3, P4, P5 are single d-amino undecanoic acid or l-amino undecanoic acid or single d-8-amino octanoic acid or l-8-Aminoctanoic acid; P6 is d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, (d-Ser-d-Tyr) or (d-Ser-d-Phe); and P7, P8 , P9, P10, P11, At least three of P12 are d-Arg or 1-Arg or d-Lys or 1-Lys, and the rest are either amino acid or absent.

在另一實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12(SEQ ID NO：35)；P12、P11、P 10、P9、P8、P7、P6、P5、P4、P3、P2、P1(SEQ ID NO：36)；P12、P11、P10、P6、P9、P4、P7、P2、P1(SEQ ID NO：37)；或P1、P2、P7、P4、P9、P6、P10、P11、P12(SEQ ID NO：38)；其中P1係d-Cha或l-Cha，或d-Nal(2)或l-Nal(2)；P2係d-(Phe-2,3,4,5,6-F)或l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)或l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)；及P7、P8、P9、P10、P11、P12中之至少三者係d-Arg或l-Arg，且其餘為任一胺基酸或不存在；P3係d-絲胺酸或l-絲胺酸；P4係d-色胺酸或l-色胺酸；P5係d-絲胺酸或l-絲胺酸或d-天冬醯胺或l-天冬醯胺；P6係d-Bpa或l-Bpa、d-Phe或l-Phe4NO2、(d-或l-Ser-d-Tyr或l-Tyr)或(d-或l-Ser-d-Phe或l-Phe)。 In another embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12 (SEQ ID NO: 35); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 36); P12, P11, P10, P6, P9, P4, P7, P2, P1 (SEQ ID NO: 37); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO: 38); wherein P1 is d-Cha or l-Cha, or d-Nal(2) or l-Nal(2); P2 is d-(Phe-2,3,4,5,6-F) or l-(Phe-2,3,4,5,6-F), d-(Phe- 3,4,5F) or l-(Phe-3,4,5F), d-(Phe-4CF3) or l-(Phe-4CF3); and at least one of P7, P8, P9, P10, P11, P12 The three are d-Arg or l-Arg, and the rest are any amino acid or absent; P3 is d-serine or l-serine; P4 is d-tryptophan or l-tryptophan ; P5 is d-serine or l-serine or d-asparagine or l-asparagine; P6 is d-Bpa or l-Bpa, d-Phe or l-Phe4NO2, (d- or l-Ser-d-Tyr or l-Tyr) or (d- or l-Ser-d-Phe or l-Phe).

在又一實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、P4、P5、P6(SEQ ID NO：39)或P6、P5、P4、P3、P2、P1(SEQ ID NO：40)；其中P1係d-Cha或l-Cha，或d-Nal(2)或l-Nal(2)；P2係(d-Phe-2,3,4,5,6-F或l-Phe-2,3,4,5,6-F)、(d-Phe-3,4,5F或l-Phe-3,4,5F)或(d-Phe-4CF3或l-Phe-4CF3)；P3係d-Ser或l-Ser；P4係d-Trp或l-Trp；P5係d-Ser或l-Ser；P6係d-Bpa或l-Bpa，或(d-或l-Ser-d-Tyr或l-Tyr)。 In yet another embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6 (SEQ ID NO: 39) or P6, P5, P4, P3, P2, P1 (SEQ ID NO: 40); where P1 is d-Cha or l-Cha, or d-Nal(2) or l-Nal(2); P2 is (d-Phe-2,3,4,5,6-F or l-Phe-2,3,4,5,6-F), (d-Phe-3,4,5F or l-Phe-3,4,5F) or (d-Phe-4CF3 or l-Phe- 4CF3); P3 is d-Ser or l-Ser; P4 is d-Trp or l-Trp; P5 is d-Ser or l-Ser; P6 is d-Bpa or l-Bpa, or (d- or l- Ser-d-Tyr or l-Tyr).

在另一實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、P4、P5、P6(SEQ ID NO：41)；P6、P5、P4、P3、P2、P1(SEQ ID NO：42)；P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12(SEQ ID NO：43)； P1、P2、P3、P4、P5、P6、P12、P11、P10、P9、P8、P7(SEQ ID NO：44)；P6、P5、P4、P3、P2、P1、P7、P8、P9、P10、P11、P12(SEQ ID NO：45)；P6、P5、P4、P3、P2、P1、P12、P11、P10、P9、P8、P7(SEQ ID NO：46)；P7、P8、P9、P10、P11、P12、P1、P2、P3、P4、P5、P6(SEQ ID NO：47)；P7、P8、P9、P10、P11、P12、P6、P5、P4、P3、P2、P1(SEQ ID NO：48)；P12、P11、P10、P9、P8、P7、P1、P2、P3、P4、P5、P6(SEQ ID NO：49)；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1(SEQ ID NO：50)；P12、P11、P6、P9、P8、P7、P2、P1(SEQ ID NO：51)；P12、P11、P10、P6、P9、P4、P7、P2、P1(SEQ ID NO：52)；P1、P2、P7、P8、P9、P6、P11、P12(SEQ ID NO：53)；或P1、P2、P7、P4、P9、P6、P10、P11、P12(SEQ ID NO：54)；其中P1係d-Cha或l-Cha，或d-Nal(2)或l-Nal(2)；P2係(d-Phe-2,3,4,5,6-F或l-Phe-2,3,4,5,6-F)、(d-Phe-3,4,5F或l-Phe-3,4,5F)或(d-Phe-4CF3或l-Phe-4CF3)；P3係任一胺基酸(例如，d-Ser或l-Ser，或d-Pro或l-Pro)；P4係d-Trp或l-Trp；P5係任一胺基酸(例如，d-Ser或l-Ser)；P7係d-Arg或l-Arg；P8係d-Arg或l-Arg；P9係d-Arg或l-Arg；P10係d-Gln或l-Gln或d-Arg或l-Arg；P11係d-Arg或l-Arg；P12係d-Arg或l-Arg；P6係d-Bpa或l-Bpa或(d-或l-Ser-d-Tyr或l-Tyr)。 In another embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6 (SEQ ID NO: 41); P6, P5, P4, P3, P2, P1 (SEQ ID NO: 41); NO: 42); P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12 (SEQ ID NO: 43); P1, P2, P3, P4, P5, P6, P12, P11, P10, P9, P8, P7 (SEQ ID NO: 44); P6, P5, P4, P3, P2, P1, P7, P8, P9, P10 , P11, P12 (SEQ ID NO: 45); P6, P5, P4, P3, P2, P1, P12, P11, P10, P9, P8, P7 (SEQ ID NO: 46); P7, P8, P9, P10 , P11, P12, P1, P2, P3, P4, P5, P6 (SEQ ID NO: 47); P7, P8, P9, P10, P11, P12, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 48); P12, P11, P10, P9, P8, P7, P1, P2, P3, P4, P5, P6 (SEQ ID NO: 49); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 50); P12, P11, P6, P9, P8, P7, P2, P1 (SEQ ID NO: 51); P12, P11, P10, P6, P9, P4, P7, P2, P1 (SEQ ID NO:52); P1, P2, P7, P8, P9, P6, P11, P12 (SEQ ID NO:53); or P1, P2, P7, P4, P9, P6 , P10, P11, P12 (SEQ ID NO: 54); wherein P1 is d-Cha or l-Cha, or d-Nal(2) or l-Nal(2); P2 is (d-Phe-2,3 ,4,5,6-F or l-Phe-2,3,4,5,6-F), (d-Phe-3,4,5F or l-Phe-3,4,5F) or (d -Phe-4CF3 or l-Phe-4CF3); P3 is any amino acid (for example, d-Ser or l-Ser, or d-Pro or l-Pro); P4 is d-Trp or l-Trp; P5 is any amino acid (for example, d-Ser or l-Ser); P7 is d-Arg or l-Arg; P8 is d-Arg or l-Arg; P9 is d-Arg or l-Arg; P10 Department d-Gln or l-Gln or d-Arg or l-Arg; P11 department d-Arg or l-Arg; P12 department d-Arg or l-Arg; P6 department d-Bpa or l-Bpa or (d- or l-Ser-d-Tyr or l-Tyr).

在另一實施例中，鄰接肽或擬肽序列包括以下結構：P1、P2、P3、P4、P5、P6、P7、P8、P9、P10、P11、P12(SEQ ID NO：55)；P12、P11、P10、P9、P8、P7、P6、P5、P4、P3、P2、P1(SEQ ID NO：56)；P12、P11、P10、P6、P9、P4、P7、P2、P1(SEQ ID NO：57)；或P1、P2、P7、P4、P9、P6、P10、P11、P12(SEQ ID NO：58)；其中P1係d-Cha或l-Cha或d-Nal(2)或l-Nal(2)；P2係(d-Phe-2,3,4,5,6-F或l-Phe-2,3,4,5,6-F)；P3 係d-Ser或l-Ser；P4係d-Trp或l-Trp；P5係d-Ser或l-Ser；P7係d-Arg或l-Arg；P8係d-Arg或l-Arg；P9係d-Arg或l-Arg；P10係d-Gln或l-Gln或d-Arg或l-Arg；P11係d-Arg或l-Arg；P12係d-Arg或l-Arg；P6係d-Bpa或l-Bpa或(d-或l-Ser-d-Tyr或l-Tyr)。 In another embodiment, the contiguous peptide or peptidomimetic sequence comprises the following structures: P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12 (SEQ ID NO: 55); P12, P11, P10, P9, P8, P7, P6, P5, P4, P3, P2, P1 (SEQ ID NO: 56); P12, P11, P10, P6, P9, P4, P7, P2, P1 (SEQ ID NO :57); or P1, P2, P7, P4, P9, P6, P10, P11, P12 (SEQ ID NO: 58); wherein P1 is d-Cha or l-Cha or d-Nal(2) or l- Nal(2); P2 line (d-Phe-2,3,4,5,6-F or l-Phe-2,3,4,5,6-F); P3 D-Ser or l-Ser; P4 d-Trp or l-Trp; P5 d-Ser or l-Ser; P7 d-Arg or l-Arg; P8 d-Arg or l-Arg; P9 Department d-Arg or l-Arg; P10 department d-Gln or l-Gln or d-Arg or l-Arg; P11 department d-Arg or l-Arg; P12 department d-Arg or l-Arg; P6 department d -Bpa or 1-Bpa or (d- or 1-Ser-d-Tyr or 1-Tyr).

在再其他實施例中，鄰接肽或擬肽序列包括以下結構：(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：99)；(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：100)；(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO：59)；(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：60)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：61)；(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO：62)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO：63)；(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO：64)；(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO：65)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg) (d-Arg)(SEQ ID NO：66)；(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：67)；(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO：68)；(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：69)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(SEQ ID NO：70)；(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：71)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO：72)；(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：73)；(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO：74)；(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：75)；或(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO：76)。 In yet other embodiments, the contiguous peptide or peptidomimetic sequence comprises the following structure: (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5, 6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 99); (d- Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d- Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO: 100); (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d- Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)( SEQ ID NO:59); (d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d- Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO:60); (d-Cha)(d-Phe-2,3 ,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Gln)( d-Arg)(d-Arg)(SEQ ID NO: 61); (d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d- Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa) (SEQ ID NO: 62); (d- Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)( d-Gln)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO: 63); (d-Arg)(d-Arg)(d-Gln)(d-Arg)(d- Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)( SEQ ID NO: 64); (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3 ,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO:65); (d-Cha)(d-Phe-2,3 ,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Arg)( d-Arg) (d-Arg)(SEQ ID NO:66); (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d -Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 67); (d-Bpa)(d -Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg) (d-Arg)(d-Arg)(d-Arg)(SEQ ID NO: 68); (d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d -Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO:69); (d-Cha)(d-Phe-2,3,4,5 ,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(SEQ ID NO: 70); (d-Arg)(d -Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:71); (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa) (d-Arg)(d-Arg)(d-Arg)(SEQ ID NO:72); (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d -Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 73); (d-Cha)(d -Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg) (d-Arg)(SEQ ID NO:74); (d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d -Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO:75); or (d-Cha)(d-Phe-2,3,4,5,6-F )(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg) (SEQ ID NO: 76).

在再其他實施例中，鄰接肽或擬肽序列包括以下結構：(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：77)。 In yet other embodiments, the contiguous peptide or peptidomimetic sequence comprises the following structure: (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5, 6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 77).

本發明肽及擬肽視情況含有poly-lys及/或arg序列以便輔助穿過細胞膜。由於其他胺基酸序列(例如，HIV tat，細胞表面受體/蛋白質等之配體)能穿過膜且其他分子可用於促進G2廢除性肽及擬肽(例如，脂質體、膠束及其他脂質分子、病毒及其他載體、電穿孔等)(包括可選的poly-lys及/或poly-arg序列)進入細胞。因此，在其他實施例中，肽及擬肽不具有輔助細胞進入之poly-lys及/或arg序列。舉例而言，在兩個具體實施例中，輔助細胞膜穿過之具有poly-lys/arg序列之最小序列包括P6、P5、P4、P3、P2、P1，例如d-Bpa、d-Ser、d-Trp、d-Ser、d-Phe-2,3,4,5,6F、d-Cha(SEQ ID NO：101)；及d-Tyr、d-Ser、d-Pro、d-Trp、d-Ser、d-Phe-2,3,4,5,6F、d-Cha(SEQ ID NO：102)。在兩個其他具體實施例中，輔助細胞膜穿過之沒有poly-lys/arg序列之最小序列包括(例如)d-Bpa、d-Cys、d-Trp、d-Ser、d-Phe-2,3,4,5,6F、d-Cha、d-Cys(SEQ ID NO：103)；及d-Tyr、d-Cys、d-Pro、d-Trp、d-Ser、d-Phe-2,3,4,5,6F、d-Cha、d-Cys(SEQ ID NO：104)；Cys殘基視情況經環化。 The peptides and peptidomimetics of the invention optionally contain poly-lys and/or arg sequences to facilitate passage through cell membranes. Since other amino acid sequences (e.g., HIV tat, ligands for cell surface receptors/proteins, etc.) and other lipid molecules, viruses and other vectors, electroporation, etc.) (including optional poly-lys and/or poly-arg sequences) into cells. Thus, in other embodiments, the peptides and peptidomimetics do not have poly-lys and/or arg sequences that facilitate cell entry. For example, in two specific embodiments, the minimum sequence with poly-lys/arg sequence to help cell membrane pass includes P6, P5, P4, P3, P2, P1, such as d-Bpa, d-Ser, d - Trp, d-Ser, d-Phe-2,3,4,5,6F, d-Cha (SEQ ID NO: 101); and d-Tyr, d-Ser, d-Pro, d-Trp, d -Ser, d-Phe-2,3,4,5,6F, d-Cha (SEQ ID NO: 102). In two other specific embodiments, the minimal sequence without poly-lys/arg sequences for cell membrane passage includes, for example, d-Bpa, d-Cys, d-Trp, d-Ser, d-Phe-2, 3,4,5,6F, d-Cha, d-Cys (SEQ ID NO: 103); and d-Tyr, d-Cys, d-Pro, d-Trp, d-Ser, d-Phe-2, 3,4,5,6F, d-Cha, d-Cys (SEQ ID NO: 104); Cys residues are optionally cyclized.

如所論述，本發明化合物單獨具有抗細胞增殖活性或G2廢除活性。可藉由將該等本發明化合物與其他藥劑(例如T細胞活化劑及/或免疫查核點抑制劑)組合來增加抗細胞增殖活性。可在方法或組合組合物中將T細胞活化劑與肽及擬肽組合。或者，可在方法或組合組合物中將免疫查核點抑制劑與肽及擬肽組合。此外，可在方法或組合組合物中將T細胞活化劑及免疫查核點抑制劑與肽及擬肽組合。因此，本發明進一步提供包括本發明化合物(例如，肽或擬肽序列)及T細胞活化劑及/或免疫查核點抑制劑之組合物，以及該等組合(例如本發明化合物(例如，肽或擬肽序列)單獨或與T細胞活化劑及/或免疫查核點抑制劑組合投與)之方法及用途。 As discussed, the compounds of the present invention alone possess anti-cell proliferative activity or G2 abrogating activity. Anti-cell proliferative activity can be increased by combining the compounds of the invention with other agents such as T cell activators and/or immune checkpoint inhibitors. T cell activators can be combined with peptides and peptidomimetics in methods or in combination compositions. Alternatively, immune checkpoint inhibitors can be combined with peptides and peptidomimetics in methods or combined compositions. In addition, T cell activators and immune checkpoint inhibitors can be combined with peptides and peptidomimetics in methods or in combination compositions. Accordingly, the invention further provides compositions comprising a compound of the invention (e.g., a peptide or peptidomimetic sequence) and a T cell activator and/or an immune checkpoint inhibitor, as well as combinations of such (e.g., a compound of the invention (e.g., a peptide or peptidomimetic sequences) alone or in combination with T cell activators and/or immune checkpoint inhibitors).

活化劑通常係降低、減少或抑制靶標分子之一或多種活性之「拮抗劑」。拮抗劑可藉由使由配體活化之細胞失能或殺死該等細胞及/或藉由在配體結合至受體後干擾受體或配體結合或活化或信號轉導，而干擾配體至受體或受體至配體之結合。拮抗劑可能但不一定完全阻斷相互作用或可實質上降低、減少或抑制該等相互作用。 An activator is generally an "antagonist" that reduces, diminishes or inhibits one or more activities of target molecules. Antagonists can interfere with ligand activation by disabling or killing cells activated by the ligand and/or by interfering with receptor or ligand binding or activation or signal transduction after ligand binding to the receptor. body to Receptor or Receptor to Ligand Binding. An antagonist may, but need not, completely block an interaction or may substantially reduce, diminish or inhibit such an interaction.

T細胞活化劑之靶標之具體實例包括CD28、OX40、GITR、CD137、CD27及HVEM。T細胞活化劑包括(但不限於)結合至CD28、OX40、GITR、CD137、CD27及/或HVEM之配體及抗體。 Specific examples of targets for T cell activators include CD28, OX40, GITR, CD137, CD27, and HVEM. T cell activators include, but are not limited to, ligands and antibodies that bind to CD28, OX40, GITR, CD137, CD27 and/or HVEM.

抗CD28抗體之特定非限制性實例係TGN1412(Chia-Huey Lin等人，(2004-11-16)Blood(ASH Annual Meeting Abstracts)104(11)：Abstract 2519；Suntharalingam等人，(2006)N Engl J Med 355：1018-1028)。 A specific non-limiting example of an anti-CD28 antibody is TGN1412 (Chia-Huey Lin et al., (2004-11-16) Blood (ASH Annual Meeting Abstracts) 104(11): Abstract 2519; Suntharalingam et al., (2006) N Engl J Med 355:1018-1028).

OX40抗體之特定非限制性實例包括MEDI6469、MEDI0562及MEDI6383(國際專利申請案PCT/US2015/023434、國際公開案WO/2015/153514)。OX40配體之特定非限制性實例係OX40L(美國專利第7291331號) Specific non-limiting examples of OX40 antibodies include MEDI6469, MEDI0562, and MEDI6383 (International Patent Application PCT/US2015/023434, International Publication WO/2015/153514). A specific non-limiting example of an OX40 ligand is OX40L (US Patent No. 7291331)

抗GITR抗體之特定非限制性實例包括TRX518(GITR,Inc.,先前由Tolerx研發)(Tolerx Inc.,(2007)Expert Opin Ther Patents 17：567-575；Ascierto PA等人，(2010)；亦參見WO/2006/105021及WO/2009/009116。 Specific non-limiting examples of anti-GITR antibodies include TRX518 (GITR, Inc., previously developed by Tolerx) (Tolerx Inc., (2007) Expert Opin Ther Patents 17:567-575; Ascierto PA et al., (2010); also See WO/2006/105021 and WO/2009/009116.

抗CD137(41BB)抗體之特定非限制性實例包括烏瑞魯單抗(Urelumab)(BMS-663513)(Bristol-Myers Squibb)(Logan,T.等人，(2008),Journal of Immunotherapy，第31卷，第9期，第956頁)及PF-05082566(PF-2566,Pfizer)(例如，如PCT/IB2011/053761(WO/2012/032433)中所闡述)。 Specific non-limiting examples of anti-CD137(41BB) antibodies include Urelumab (BMS-663513) (Bristol-Myers Squibb) (Logan, T. et al., (2008), Journal of Immunotherapy, p. 31 Vol. 9, p. 956) and PF-05082566 (PF-2566, Pfizer) (eg, as described in PCT/IB2011/053761 (WO/2012/032433)).

抗CD27抗體之特定非限制性實例係CDX-1127(Varlilumab,Celldex Therapeutics)(Vitale LA等人(2012)Clin Cancer Res.18(14)：3812-21)。 A specific non-limiting example of an anti-CD27 antibody is CDX-1127 (Varlilumab, Celldex Therapeutics) (Vitale LA et al. (2012) Clin Cancer Res. 18(14):3812-21).

抗HVEM抗體之特定非限制性實例係可自闡述於美國專利第8349320 號中之雜交瘤獲得之單株抗體(以編號CNCM 1-4752儲存於Collection Nationale de Cultures de Microorganismes,Institut Pasteur,25 rue du Docteur Roux,75724 Paris Cedex 15,France處)。 Specific non-limiting examples of anti-HVEM antibodies can be found in U.S. Pat. No. 8,349,320 The monoclonal antibody obtained from the hybridoma No. (deposited at Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France under the number CNCM 1-4752).

免疫查核點抑制劑之靶標之具體實例包括CTLA-4、PD1、PD-L1、PDL2、VISTA、TIM3、LAG-3及BTLA。免疫查核點抑制劑包括(但不限於)結合至CTLA-4、PD1、PD-L1、PDL2、VISTA、TIM3、LAG-3及/或BTLA之配體及抗體。 Specific examples of targets of immune checkpoint inhibitors include CTLA-4, PD1, PD-L1, PDL2, VISTA, TIM3, LAG-3, and BTLA. Immune checkpoint inhibitors include, but are not limited to, ligands and antibodies that bind to CTLA-4, PD1, PD-L1, PDL2, VISTA, TIM3, LAG-3 and/or BTLA.

抗PD-1抗體及相關分子之特定非限制性實例包括尼沃魯單抗(Nivolumab)(BMS-936558/MDX-1106/ONO-4538)(Brahmer JR等人，(2010)J Clin Oncol.28(19)：3167-75)；蘭布魯珠單抗(Lambrolizumab)(別名：MK3475(Merck))；派姆單抗(Pembrolizumab)(Keytruda)(美國專利第8952136號)；匹利珠單抗(Pidilizumab)(CT-011,CureTech)(Westin JR等人，(2014)Lancet Oncol.15(1)：69-77)；AMP-514(MedImmune/AZ，別名：MEDI0680)(WO/2012/145493)；及AMP-224(GSK，別名：B7-DCIg(在WO2010/027827及WO2011/066342中闡述之PDL2-Fc融合可溶性受體))。 Specific non-limiting examples of anti-PD-1 antibodies and related molecules include Nivolumab (BMS-936558/MDX-1106/ONO-4538) (Brahmer JR et al., (2010) J Clin Oncol. 28 (19): 3167-75); Lambrolizumab (alias: MK3475 (Merck)); Pembrolizumab (Keytruda) (US Patent No. 8952136); Pilizumab (Pidilizumab) (CT-011, CureTech) (Westin JR et al., (2014) Lancet Oncol. 15(1):69-77); AMP-514 (MedImmune/AZ, alias: MEDI0680) (WO/2012/145493 ); and AMP-224 (GSK, alias: B7-DCIg (PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342)).

PD-1拮抗劑分子之特定非限制性實例包括AUNP12(Aurigene及Pierre Fabre Pharmaceuticals；美國專利公開案第US20110318373號)。 Specific non-limiting examples of PD-1 antagonist molecules include AUNP12 (Aurigene and Pierre Fabre Pharmaceuticals; US Patent Publication No. US20110318373).

抗PD-L1抗體之特定非限制性實例包括BMS-936559(Bristol-Myers Squibb)(WO/2007/005874)；MPDL3280A(Genentech)(RG7446；WO/2010/077634)；MedI-4736(Medimmune，先前Pfizer),MSB0010718C(EMD Serono)(WO/2013/79174)；及MDX-1105(WO/2007/005874)。 Specific non-limiting examples of anti-PD-L1 antibodies include BMS-936559 (Bristol-Myers Squibb) (WO/2007/005874); MPDL3280A (Genentech) (RG7446; WO/2010/077634); MedI-4736 (Medimmune, previously Pfizer), MSB0010718C (EMD Serono) (WO/2013/79174); and MDX-1105 (WO/2007/005874).

抗CTLA-4抗體之特定非限制性實例包括伊匹單抗 (Ipilimumab)(Bristol-Myers Squibb,Yervoy®)(國際申請案第PCT/US1999/28739號(作為WO/2000/32231公開)、美國專利第5811097號、第5855887號、第6051227號，、及第6207156號))；及曲美目單抗(Tremelimumab)(先前為CP-675,206)(Medimmune，先前Pfizer)(Calabrò L等人，(2013)Lancet Oncol.14(11)：1104-11；及Camacho,LH等人，(2004)Journal Of Clinical Oncology，增刊(S)，第22卷，第14期，第164S-164S頁)。 Specific non-limiting examples of anti-CTLA-4 antibodies include ipilimumab (Ipilimumab) (Bristol-Myers Squibb, Yervoy®) (International Application No. PCT/US1999/28739 (published as WO/2000/32231), U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, and 6207156)); and Tremelimumab (formerly CP-675,206) (Medimmune, formerly Pfizer) (Calabrò L et al., (2013) Lancet Oncol.14(11):1104-11; and Camacho , LH et al., (2004) Journal Of Clinical Oncology, Suppl (S), Vol. 22, No. 14, pp. 164S-164S).

抗LAG-3抗體之特定非限制性實例包括BMS-986016(Bristol-Myers Squibb)(WO/2010/19570,WO/2014/08218)；IMP-731或IMP-321(WO/2008/132601,WO/2009/44273)；及如(國際專利申請案第PCT/US2015/020474,(WO/2015/138920))中所闡述之其他抗LAG3抗體。 Specific non-limiting examples of anti-LAG-3 antibodies include BMS-986016 (Bristol-Myers Squibb) (WO/2010/19570, WO/2014/08218); IMP-731 or IMP-321 (WO/2008/132601, WO /2009/44273); and other anti-LAG3 antibodies as described in (International Patent Application No. PCT/US2015/020474, (WO/2015/138920)).

抗TIM3抗體之特定非限制性實例闡述於美國專利第8841418號及美國專利公開案第20150086574號中。 Specific non-limiting examples of anti-TIM3 antibodies are described in US Patent No. 8841418 and US Patent Publication No. 20150086574.

抗VISTA(B7-H5)抗體之特定非限制性實例闡述於WO 2014190356中。 Specific non-limiting examples of anti-VISTA (B7-H5) antibodies are described in WO 2014190356.

抗BTLA抗體之特定非限制性實例闡述於WO2008076560、US8563694、US20120064096、US20100172900中。 Specific non-limiting examples of anti-BTLA antibodies are described in WO2008076560, US8563694, US20120064096, US20100172900.

術語「抗體」係指經由重鏈及輕鏈可變結構域(分別表示為V_H及V_L)結合至另一分子(抗原)之蛋白質。「抗體」係指任一多株或單株免疫球蛋白分子或其混合物(例如IgM、IgG、IgA、IgE、IgD)。抗體屬任一抗體類別或亞類。IgG之實例性亞類係IgG₁、IgG₂、IgG₃及IgG₄。 The term "antibody" refers to a protein that binds to another molecule (antigen) through the variable domains of the heavy and light chains (denoted _VH and _VL , respectively). "Antibody" refers to any polyclonal or monoclonal immunoglobulin molecule or mixture thereof (eg, IgM, IgG, IgA, IgE, IgD). Antibodies belong to any antibody class or subclass. Exemplary subclasses of IgG are IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ .

術語「單株」在提及抗體使用時係指基於單一純系(包括任一真核、原核或噬菌體純系)、自其獲得或源於其之抗體。因此，「單株」抗體在本文係在結構上而非在產生其之方法上來定義。 The term "monoclonal" when used in reference to an antibody refers to an antibody that is based on, obtained from, or derived from a single clonal line (including any eukaryotic, prokaryotic, or phage clonal line). Thus, "monoclonal" antibodies are defined herein by their structure and not by the method by which they are produced.

抗體包括κ輕鏈或λ輕鏈序列(如在天然抗體中之全長)、其混合物(即，κ及λ鏈序列之融合)及其子序列/片段。天然抗體分子含有兩條κ及兩條λ輕鏈。κ輕鏈與λ輕鏈之間之主要差異在於恆定區之序列。 Antibodies include kappa or lambda light chain sequences (full length as in native antibodies), mixtures thereof (ie, fusions of kappa and lambda chain sequences), and subsequences/fragments thereof. Natural antibody molecules contain two kappa and two lambda light chains. The main difference between kappa and lambda light chains is the sequence of the constant region.

包括重(H)鏈及/或輕(L)鏈或重(H)鏈或輕(L)鏈之片段或由其組成之抗體可包括單一H鏈或L鏈或單一H鏈或L鏈片段，或複數條(2條、3條、4條或更多條)重(H)鏈及/或輕(L)鏈，或重(H)鏈及/或輕(L)鏈之複數個片段。抗體或片段可能但不需要包括2條重(H)鏈及2條輕(L)鏈。抗體或其片段可與其他抗體、其片段、重(H)鏈、輕(L)鏈或不同於抗體重(H)鏈或輕(L)鏈之多肽一起為寡聚(更高階或價)形式，例如三聚體、四聚體、五聚體、六聚體、七聚體及等等。 An antibody comprising or consisting of a heavy (H) chain and/or a light (L) chain or a fragment of a heavy (H) chain or a light (L) chain may comprise a single H chain or L chain or a single H chain or L chain fragment , or a plurality (2, 3, 4 or more) of heavy (H) chains and/or light (L) chains, or multiple fragments of heavy (H) chains and/or light (L) chains . An antibody or fragment may, but need not, comprise 2 heavy (H) chains and 2 light (L) chains. Antibodies or fragments thereof may be oligomeric (higher order or valence) with other antibodies, fragments thereof, heavy (H) chains, light (L) chains, or polypeptides different from antibody heavy (H) or light (L) chains Forms such as trimers, tetramers, pentamers, hexamers, heptamers, and the like.

術語「融合」或「嵌合體」及其語法變化形式在用於提及序列時意味著該序列含有一或多個基於兩種或更多種不同蛋白質、源於其或自其獲得或分離之部分。亦即例如，序列之一部分可基於或來自一種具體蛋白質，且序列之另一部分可基於或來自不同的具體蛋白質。因此，融合或嵌合多肽係其中多肽之不同部分具有不同蛋白質起源之分子。 The term "fusion" or "chimera" and grammatical variations thereof when used in reference to a sequence means that the sequence contains one or more proteins based on, derived from, obtained or isolated from two or more different proteins. part. That is, for example, one part of the sequence may be based on or derived from one specific protein and another part of the sequence may be based on or derived from a different specific protein. Thus, fusion or chimeric polypeptides are molecules in which different parts of the polypeptide have different protein origins.

抗體包括包括哺乳動物、人類、人類化及靈長類化序列。術語「人類」在提及抗體時意味著胺基酸序列係完全人類的。因此，「人類抗體」係指具有人類免疫球蛋白胺基酸序列(亦即特異性結合至靶標之人類重鏈及輕鏈可變及恆定區)之抗體。亦即，所有抗體胺基酸皆為人類的或可能或確實存在於人類抗體中。 Antibodies include mammalian, human, humanized and primatized sequences. The term "human" in reference to an antibody means that the amino acid sequence is fully human. Thus, "human antibody" refers to an antibody having human immunoglobulin amino acid sequences, ie, human heavy and light chain variable and constant regions that specifically bind to a target. That is, all antibody amino acids are human or can or do exist in human antibodies.

可藉由利用可能或確實存於人類抗體中之胺基酸殘基取代非人類胺基酸殘基將為非人類抗體之抗體製成完全人類抗體。胺基酸殘基存在於人類抗體中，CDR區圖及人類抗體共有殘基為業內已知(例如，參見Kabat, Sequences of Proteins of Immunological Interest，第4版US Department of Health and Human Services.Public Health Service(1987)；及Chothia及Lesk J.Mol.Biol.186：651(1987))。基於22個已知人類V_H III序列之調查之人類V_H亞組III之共有序列及基於30個已知人類κ I序列之調查之人類V_L κ鏈亞組I之共有序列闡述於Padlan Mol.Immunol.31：169(1994)及Padlan Mol.Immunol.28：489(1991)中。因此，人類抗體包括其中一或多個胺基酸殘基經存於另一人類抗體中之一或多個胺基酸取代之抗體。 Fully human antibodies can be made for antibodies that would be non-human antibodies by substituting non-human amino acid residues with amino acid residues that could or do exist in human antibodies. Amino acid residues are present in human antibodies, CDR region maps and consensus residues for human antibodies are known in the art (see, for example, Kabat, Sequences of Proteins of Immunological Interest , 4th edition US Department of Health and Human Services. Public Health Service (1987); and Chothia and Lesk J. Mol. Biol. 186:651 (1987)). The consensus sequence of human _VH subgroup III based on a survey of 22 known human _VHIII sequences and the consensus sequence of human _VL κ chain subgroup I based on a survey of 30 known human κI sequences are described in Padlan Mol Immunol. 31:169 (1994) and Padlan Mol. Immunol. 28:489 (1991). Thus, human antibodies include antibodies in which one or more amino acid residues are substituted with one or more amino acid residues found in another human antibody.

術語「人類化」在用於提及抗體時，意味著該抗體之胺基酸序列具有帶有一或多個特異性結合至受體人類免疫球蛋白分子之期望靶標之決定區(CDR)之非人類胺基酸殘基(例如，小鼠、大鼠、山羊、兔、非人類靈長類動物等)，及一或多個在Fv框架區(FR)中且位於CDR兩側之胺基酸殘基之人類胺基酸殘基。免疫球蛋白之人類框架區殘基可經相應的非人類殘基替代。因此，人類框架區中之殘基可經來自非人類CDR供體抗體之相應殘基取代以改變、通常改良(例如)抗原親和性或特異性。另外，人類化抗體可包括在人類抗體及供體CDR或框架序列中皆未發現之殘基。舉例而言，可預測在人類抗體或供體非人類抗體中未發現之特定位置處之框架取代可改良該位置處人類抗體之結合親和性或特異性。 The term "humanized" when used in reference to an antibody means that the amino acid sequence of the antibody has non-identical regions (CDRs) with one or more determining regions (CDRs) that specifically bind to the desired target of the recipient human immunoglobulin molecule. Human amino acid residues (e.g., mouse, rat, goat, rabbit, non-human primate, etc.), and one or more amino acids in the Fv framework region (FR) flanking the CDRs Residues are human amino acid residues. Human framework region residues of the immunoglobulin may be substituted by corresponding non-human residues. Thus, residues in the human framework regions may be substituted with corresponding residues from a non-human CDR donor antibody to alter, often improve, for example, antigen affinity or specificity. Additionally, humanized antibodies may include residues that are found neither in human antibodies nor in the donor CDR or framework sequences. For example, a framework substitution at a particular position not found in the human antibody or donor non-human antibody can be predicted to improve the binding affinity or specificity of the human antibody at that position.

基於分子建模之抗體框架及CDR取代為業內已知，例如藉由對CDR及框架殘基之相互作用建模以鑑別於抗原結合而言重要之框架殘基，並進行序列比較以鑑別特定位置處之不尋常的框架殘基(例如，參見美國專利第5,585,089號；及Riechmann等人，Nature 332：323(1988))。稱作「靈長類化」之抗體在如本文所使用之「人類化」之含義內，只是受體人類免疫球蛋白分子及框架區胺基酸殘基可為除任一人類殘基以外之任一靈長類動物胺基酸殘基(例如，人猿、長臂猿、大猩猩、黑猩猩、獼猴)。 Antibody framework and CDR substitutions based on molecular modeling are known in the art, for example by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and performing sequence comparisons to identify specific positions Unusual framework residues are included (see, eg, US Patent No. 5,585,089; and Riechmann et al., Nature 332:323 (1988)). Antibodies referred to as "primatized" are within the meaning of "humanized" as used herein, except that the receptor human immunoglobulin molecule and framework region amino acid residues may be other than any human residues. Any primate amino acid residue (eg, orangutan, gibbon, gorilla, chimpanzee, macaque).

抗體可使用業內已知之各種技術來人類化，包括(例如)CDR移植(EP 239,400；W091/09967；美國專利第5,225,539號；第5,530,101號；及第5,585,089號)、鑲飾或表面重修(EP 592,106；EP 519,596；Padlan,Molecular lmmunol.28：489(1991)；Studnicka等人，Protein Engineering 7：805(1994)；Roguska.等人，Proc.Nat’l.Acad.Sci.USA 91：969(1994))及鏈改組(美國專利第5,565,332號)。可使用人類共有序列(Padlan,Mol.Immunol.31：169(1994)；及Padlan,Mol.Immunol.28：489(1991))來人類化抗體(Carter等人，Proc.Natl.Acad.Sci.USA 89：4285(1992)；及Presta等人，J.Immunol.151：2623(1993))。 Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting (EP 239,400; W091/09967; US Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106 EP 519,596; Padlan, Molecular lmmunol. 28:489 (1991); Studnicka et al., Protein Engineering 7:805 (1994); Roguska. et al., Proc.Nat'l.Acad.Sci.USA 91:969 (1994 )) and chain shuffling (US Patent No. 5,565,332). Antibodies can be humanized ( Carter et al . , Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993)).

產生嵌合抗體之方法為業內已知(例如，Morrison,Science 229：1202(1985)；Oi等人，BioTechniques 4：214(1986)；Gillies等人，J.Immunol.Methods 125：191(1989)；及美國專利第5,807,715號；第4,816,567號；及第4,816,397號)。用一個物種之抗體之可變結構域取代另一物種之可變結構域之嵌合抗體闡述於例如以下文獻中：Munro,Nature 312：597(1984)；Neuberger等人，Nature 312：604(1984)；Sharon等人，Nature 309：364(1984)；Morrison等人，Proc.Nat’l.Acad.Sci.USA 81：6851(1984)；Boulianne等人，Nature 312：643(1984)；Capon等人，Nature 337：525(1989)；及Traunecker等人，Nature 339：68(1989)。 Methods for generating chimeric antibodies are known in the art (e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191 (1989) and US Patent Nos. 5,807,715; 4,816,567; and 4,816,397). Chimeric antibodies in which the variable domains of antibodies from one species are substituted for variable domains from another species are described, for example, in Munro, Nature 312:597 (1984); Neuberger et al., Nature 312:604 (1984) ); Sharon et al., Nature 309:364 (1984); Morrison et al., Proc. Nat'l. Acad. Sci. USA 81:6851 (1984); Boulianne et al., Nature 312:643 (1984); Human, Nature 337:525 (1989); and Traunecker et al., Nature 339:68 (1989).

抗體包括結合至CD28、OX40、GITR、CD137、CD27、HVEM、CTLA-4、PD1、PD-L1、PDL2、VISTA、TIM3、LAG-3及/或BTLA中之一或多者雙特異性抗體。如本文所用術語「雙特異性」及其語法變化形式在提及抗體使用時意味著該抗體結合至兩個不同靶標。當靶標具有不同胺基酸序列時，將其視為係不同的。 Antibodies include bispecific antibodies that bind to one or more of CD28, OX40, GITR, CD137, CD27, HVEM, CTLA-4, PD1, PD-L1, PDL2, VISTA, TIM3, LAG-3 and/or BTLA. The term "bispecific" and its grammatical variants as used herein when used in reference to an antibody means that the antibody binds to two different targets. When targets have different amines amino acid sequences are considered to be distinct.

抗體包括子序列及片段，其係指參考抗體之功能片段或子序列，例如抗體之一或多個胺基酸缺失。抗體子序列之非限制性實例包括Fab、Fab’、F(ab’)₂、Fv、Fd、單鏈Fv(scFv)、二硫鍵連接之Fvs(sdFv)、V_L、V_H、雙價抗體((V_L-V_H)₂或(V_H-V_L)₂)、三價抗體(三價)、四價抗體(四價)、微小抗體((scF_V-C_H3)₂)、IgGdeltaCH2、scFv-Fc或(scFv)₂-Fc片段。在具體態樣中，Fab、Fab’、F(ab’)₂、Fv、Fd、單鏈Fv(scFv)、二硫鍵連接之Fvs(sdFv)、V_L、V_H、雙價抗體((V_L-V_H)₂或(V_H-V_L)₂)、三價抗體(三價)、四價抗體(四價)、微小抗體((scF_V-C_H3)₂)、IgGdeltaCH2、scFv-Fc或(scFv)₂-Fc子序列。 Antibodies include subsequences and fragments, which refer to functional fragments or subsequences of a reference antibody, eg, deletion of one or more amino acids of an antibody. Non-limiting examples of antibody subsequences include Fab, Fab', F(ab') ₂ , Fv, Fd, single chain Fv (scFv), disulfide-linked Fvs (sdFv), _VL , _VH , bivalent Antibody ((V _L -V _H ) ₂ or (V _H -V _L ) ₂ ), trivalent antibody (trivalent), tetravalent antibody (tetravalent), miniature antibody ((scF _V _-CH 3) ₂ ) , IgGdeltaCH2, scFv-Fc or (scFv) ₂ -Fc fragments. In specific aspects, Fab, Fab', F(ab') ₂ , Fv, Fd, single chain Fv (scFv), disulfide-linked Fvs (sdFv), V _L , V _H , diabody (( V _L -V _H ) ₂ or (V _H -V _L ) ₂ ), trivalent antibody (trivalent), tetravalent antibody (tetravalent), minibody ((scF _V _-CH 3) ₂ ), IgGdeltaCH2, scFv-Fc or (scFv) ₂ -Fc subsequence.

「功能子序列」或「功能片段」在提及抗體時係指保留一或多種功能或活性之至少一部分作為完整參考抗體(例如結合至靶標(例如CD28、OX40、GITR、CD137、CD27、HVEM、CTLA-4、PD1、PD-L1、PDL2、VISTA、TIM3、LAG-3及/或BTLA)之抗體)之抗體部分。 "Functional subsequence" or "functional fragment" in reference to an antibody refers to retaining at least a portion of one or more functions or activities as the intact reference antibody (e.g., binds to a target (e.g., CD28, OX40, GITR, CD137, CD27, HVEM, Antibody portions of antibodies to CTLA-4, PD1, PD-L1, PDL2, VISTA, TIM3, LAG-3 and/or BTLA).

抗體子序列(包括單鏈抗體)可包括單獨的重鏈或輕鏈可變區(例如，CDR1、CDR2或CDR3)之全部或一部分或其與以下各項中之一或多者之全部或一部分的組合：鉸鏈區、CH1、CH2及CH3結構域。亦包括重鏈或輕鏈可變區(例如，CDR1、CDR2或CDR3)與鉸鏈區、CH1、CH2及CH3結構域之任一組合之抗原-結合子序列。 Antibody subsequences (including single chain antibodies) may include all or a portion of an individual heavy or light chain variable region (e.g., CDR1, CDR2, or CDR3) or all or a portion of it in combination with one or more of Combination of: hinge region, CH1, CH2 and CH3 domains. Antigen-binding subsequences of any combination of heavy or light chain variable regions (eg, CDR1 , CDR2, or CDR3) and hinge, CH1 , CH2, and CH3 domains are also included.

亦可藉由將本發明化合物與其他藥劑(例如直接或間接造成核酸損害之治療劑)組合來進一步增加抗細胞增殖活性。亦可藉由將該等本發明化合物與抑制細胞增殖之治療劑(無論治療是否損害核酸)組合來增加抗細胞增殖活性。因此，本發明進一步提供包括本發明化合物(例如，肽或擬肽序列)及核酸損害劑之組合物及包括本發明化合物(例如，肽或擬肽序列)及抗增殖劑之組合物。 Anti-cell proliferative activity can also be further increased by combining the compounds of the invention with other agents, such as therapeutic agents that directly or indirectly cause nucleic acid damage. Anti-cell proliferative activity can also be increased by combining the compounds of the invention with therapeutic agents that inhibit cell proliferation, whether or not the treatment damages nucleic acids. Accordingly, the invention further provides compositions comprising a compound of the invention (e.g., a peptide or peptidomimetic sequence) and a nucleic acid damaging agent and compositions comprising a compound of the invention (e.g., a peptide or peptidomimetic sequence) and an anti-inflammatory agent. Composition of probiotics.

如本文所用，術語「廢除細胞週期G2查核點」、「中斷細胞週期G2查核點」、「損害細胞週期G2查核點」及其語法變化形式意味著抑制細胞以在G2查核點處使細胞週期停滯。細胞週期G2查核點經廢除之細胞展現細胞處於G2查核點之時長減少，其可在完全不存在G2查核點至在適當條件下G2查核點之持續時間減少數分鐘、數小時、數天、數週或更長之範圍內。因此，與本發明化合物接觸之細胞之G2查核點之時間長度短於在不存在該化合物下細胞通常可具有之時間長度。舉例而言，G2查核點時間長度之減少可意味著在G2中保持某一時間(例如4小時)之細胞在與本發明化合物接觸時在G2中保持小於4小時，例如3.5小時、3小時、2.5小時、2小時、1小時或更短。 As used herein, the terms "abrogate the cell cycle G2 checkpoint", "interrupt the cell cycle G2 checkpoint", "impair the cell cycle G2 checkpoint" and grammatical variations thereof mean to inhibit the cell to arrest the cell cycle at the G2 checkpoint . Cell cycle G2 checkpoint abolished cells exhibit a reduction in the length of time the cell is at the G2 checkpoint, which can be reduced by minutes, hours, days, from the complete absence of the G2 checkpoint to the duration of the G2 checkpoint under appropriate conditions. range of several weeks or longer. Thus, cells exposed to a compound of the invention have a G2 checkpoint for a shorter length of time than cells would normally have in the absence of the compound. For example, a reduction in the length of time at the G2 checkpoint may mean that cells that remain in G2 for a certain period of time, such as 4 hours, remain in G2 for less than 4 hours, such as 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1 hour or less.

如本文所用術語「細胞凋亡」係指程式性細胞死亡，及細胞生理學之相關變化，例如核酸片段化、半胱天冬酶活化等，如內頁所瞭解。術語「劇變」意指由有絲分裂過程中之錯誤引起之細胞死亡。在劇變中，存在更少特徵在於細胞凋亡(例如半胱天冬酶活化、染色體凝縮等)之特徵。 The term "apoptosis" as used herein refers to programmed cell death, and associated changes in cell physiology, such as nucleic acid fragmentation, caspase activation, etc., as understood on the inside page. The term "catastrophe" means cell death caused by errors in the mitotic process. In catastrophe, there are fewer features that are characteristic of apoptosis (eg, caspase activation, chromosome condensation, etc.).

如本文所用，術語「肽」、「多肽」及「蛋白質」可互換使用且係指兩種或更多種胺基酸藉由醯胺鍵或非醯胺等效物共價連接。本發明之肽可具有任一長度。舉例而言，肽可具有約5至100個或更多個殘基，例如長度為5至12個、12至15個、15至18個、18至25個、25至50個、50至75個、75至100個或更多個殘基。本發明之肽包括l-異構物及d-異構物及l-異構物及d-異構物之組合。肽可包括通常與蛋白質之轉譯後處理相關之修飾，例如，環化(例如，二硫鍵或醯胺鍵)、磷酸化、醣基化、羧基化、泛蛋白化、肉豆蔻基化或脂質化。 As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to two or more amino acids covalently linked by amide bonds or non-amide equivalents. The peptides of the invention can be of any length. For example, a peptide can have about 5 to 100 or more residues, e.g., 5 to 12, 12 to 15, 15 to 18, 18 to 25, 25 to 50, 50 to 75 , 75 to 100 or more residues. The peptides of the present invention include l-isomers and d-isomers and combinations of l-isomers and d-isomers. Peptides may include modifications commonly associated with post-translational processing of proteins, for example, cyclization (e.g., disulfide or amide bonds), phosphorylation, glycosylation, carboxylation, ubiquitination, myristylation, or lipid change.

本文所揭示之肽進一步包括具有胺基酸結構及功能類似物之化合物，例如，具有合成或非天然胺基酸或胺基酸類似物之擬肽，只要該模擬物具有一或多種功能或活性即可。因此，本發明化合物包括「模擬物」及「擬肽」形式。 The peptides disclosed herein further include compounds having structural and functional analogs of amino acids, e.g., peptidomimetics having synthetic or non-natural amino acids or amino acid analogs, so long as the mimetic has one or more functions or activities That's it. Accordingly, the compounds of the present invention include "mimetic" and "peptidomimetic" forms.

如本文所用，術語「模擬物」及「擬肽」係指具有與本發明肽實質上相同之結構及/或功能特徵之合成型化學化合物。模擬物可完全由合成、非天然胺基酸類似物組成，或可為包括一或多種天然肽胺基酸及一或多種非天然胺基酸類似物之嵌合分子。模擬物亦可納入任一數量之天然胺基酸保守取代，只要該等取代不破壞模擬物之活性即可。與為保守變體之本發明多肽一樣，可使用常規測試來確定模擬物是否具有必要活性，例如其具有可檢測到之細胞週期G2查核點廢除性活性。在投與個體或在細胞上接觸時，可檢測到的中斷G2細胞週期查核點之模擬物因此可具有G2查核點廢除活性。 As used herein, the terms "mimetic" and "peptidomimetic" refer to synthetic chemical compounds having substantially the same structural and/or functional characteristics as the peptides of the present invention. A mimetic may consist entirely of synthetic, non-natural amino acid analogs, or may be a chimeric molecule comprising one or more natural peptide amino acids and one or more non-natural amino acid analogs. Mimetics can also incorporate any number of natural amino acid conservative substitutions, as long as such substitutions do not destroy the activity of the mimetic. As with polypeptides of the invention that are conservative variants, routine testing can be used to determine whether a mimetic possesses the requisite activity, eg, it has detectable cell cycle G2 checkpoint abrogating activity. Mimetics that detectably interrupt the G2 cell cycle checkpoint when administered to an individual or contacted on a cell may thus have G2 checkpoint abrogating activity.

肽模擬物組合物可含有通常來自三種結構基團之非天然結構組份之任一組合：a)除天然醯胺鍵(「肽鍵」)鍵聯以外之殘基鍵聯基團；b)代替天然胺基酸殘基之非天然殘基；或c)誘導二級結構模擬(亦即誘導或穩定二級結構，例如β轉折、γ轉角、β褶疊、α螺旋構象及諸如此類)之殘基。舉例而言，當殘基中之一或多者藉由除醯胺鍵以外之化學方式接合時，多肽可表徵為模擬物。個別擬肽殘基可藉由醯胺鍵、非天然及非醯胺化學鍵其他化學鍵或偶合方式(包括(例如)戊二醛、N-羥基琥珀醯亞胺酯、雙功能馬來醯亞胺、N,N’-二環己基碳二亞胺(DCC)或N,N’-二異丙基碳二亞胺(DIC))接合。替代醯胺鍵之連接基團包括(例如)酮基亞甲基(例如，用-C(=O)-NH-替代-C(=O)-CH₂-)、胺基亞甲基(CH₂-NH)、伸乙基、烯烴(CH=CH)、醚 (CH₂-O)、硫醚(CH₂-S)、四唑(CN₄-)、噻唑、反式醯胺、硫醯胺或酯(例如，參見Spatola(1983)之Chemistry and Biochemistry of Amino Acids,Peptides and Proteins，第7卷，第267頁至第357頁，「Peptide and Backbone Modifications,」Marcel Decker,NY)。 Peptidomimetic compositions may contain any combination of non-natural structural components generally derived from three types of structural groups: a) residue linkages other than natural amide bond ("peptide bond") linkages; b) Unnatural residues that replace natural amino acid residues; or c) residues that induce secondary structure mimicry (that is, induce or stabilize secondary structure, such as β-turns, γ-turns, β-sheets, α-helical conformations, and the like) base. For example, a polypeptide can be characterized as a mimetic when one or more of the residues are joined by chemical means other than amide bonds. Individual peptidomimetic residues can be linked via amide bonds, non-natural and non-amide chemical bonds, other chemical bonds or coupling means including, for example, glutaraldehyde, N-hydroxysuccinimidyl esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC)) conjugation. Linking groups that replace amide linkages include, for example, ketomethylene (e.g., -C(=O)-NH- instead of -C(=O) _-CH2- ), aminomethylene (CH ₂ -NH), ethylidene, olefin (CH=CH), ether (CH ₂ -O), thioether (CH ₂ -S), tetrazole (CN ₄ -), thiazole, trans amide, sulfoamide Amines or esters (see, eg, Spatola (1983) Chemistry and Biochemistry of Amino Acids, Peptides and Proteins , Vol. 7, pp. 267-357, "Peptide and Backbone Modifications," Marcel Decker, NY).

如所論述，肽可因替代天然胺基酸殘基含有一或多個非天然殘基而表徵為模擬物。非天然殘基為業內已知。可用作天然胺基酸殘基之模擬物之非天然殘基之具體非限制性實例係芳香族胺基酸之模擬物，該等芳香族胺基酸包括(例如)D-萘基丙胺酸或L-萘基丙胺酸；D-苯基甘胺酸或L-苯基甘胺酸；D-2-噻吩基丙胺酸或L-2-噻吩基丙胺酸；D-1、-2、3-或4-芘基丙胺酸或L-1、-2、3-或4-芘基丙胺酸；D-3-噻吩基丙胺酸或L-3-噻吩基丙胺酸；D-(2-吡啶基)-丙胺酸或L-(2-吡啶基)-丙胺酸；D-(3-吡啶基)-丙胺酸或L-(3-吡啶基)-丙胺酸；D-(2-吡嗪基)-丙胺酸或L-(2-吡嗪基)-丙胺酸；D-(4-異丙基)-苯基甘胺酸或L-(4-異丙基)-苯基甘胺酸；D-(三氟甲基)-苯基甘胺酸；D-(三氟甲基)-苯丙胺酸；D-對-氟-苯丙胺酸；D-對-聯苯苯丙胺酸或L-對-聯苯苯丙胺酸；K-對-甲氧基-聯苯苯丙胺酸或L-對-甲氧基-聯苯苯丙胺酸；D-2-吲哚(烷基)丙胺酸或L-2-吲哚(烷基)丙胺酸；及D-烷基丙胺酸或L-烷基丙胺酸，其中烷基可經甲基、乙基、丙基、己基、丁基、戊基、異丙基、異丁基、第二異丁基(sec-isotyl)、異戊基或非酸性胺基酸取代或未經取代。可用於替代天然芳香族環之非天然胺基酸之芳香族環包括(例如)噻唑基、噻吩基、吡唑基、苯并咪唑基、萘基、呋喃基、吡咯基及吡啶基芳香族環。 As discussed, a peptide can be characterized as a mimetic by containing one or more non-natural residues in place of natural amino acid residues. Unnatural residues are known in the art. Specific non-limiting examples of non-natural residues that can be used as mimetics of natural amino acid residues are mimetics of aromatic amino acids including, for example, D-naphthylalanine or L-naphthylalanine; D-phenylglycine or L-phenylglycine; D-2-thienylalanine or L-2-thienylalanine; D-1, -2, 3 - or 4-pyrenylalanine or L-1, -2, 3- or 4-pyrenylalanine; D-3-thienylalanine or L-3-thienylalanine; D-(2-pyridine base)-alanine or L-(2-pyridyl)-alanine; D-(3-pyridyl)-alanine or L-(3-pyridyl)-alanine; D-(2-pyrazinyl )-alanine or L-(2-pyrazinyl)-alanine; D-(4-isopropyl)-phenylglycine or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D-p-biphenylalanine or L-p-biphenylalanine Phenylalanine; K-p-methoxy-biphenylalanine or L-p-methoxy-biphenylalanine; D-2-indole(alkyl)alanine or L-2-indole ( Alkyl)alanine; and D-alkylalanine or L-alkylalanine, wherein the alkyl group can be modified by methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl , the second isobutyl (sec-isotyl), isopentyl or non-acidic amino acid substituted or unsubstituted. Aromatic rings of unnatural amino acids that can be used in place of natural aromatic rings include, for example, thiazolyl, thienyl, pyrazolyl, benzimidazolyl, naphthyl, furyl, pyrrolyl, and pyridyl aromatic rings .

可藉由利用同時維持負電荷之非羧酸酯胺基酸、(膦醯基)丙胺酸及硫酸化蘇胺酸取代來生成酸性胺基酸之模擬物。亦可藉由與碳二亞胺 (R’-N-C-N-R’)(包括(例如)1-環己基-3-(2-嗎啉基-(4-乙基)碳二亞胺或1-乙基-3-(4-氮陽離子-4,4-二甲基戊基)碳二亞胺)反應來選擇性地修飾羧基側基(例如，天冬胺醯基或麩胺醯基)。亦可藉由與銨離子反應將天冬胺醯基或麩胺醯基轉化為天冬醯胺基及麩醯胺基。 Mimics of acidic amino acids can be generated by substitution with non-carboxylate amino acids, (phosphono)alanine, and sulfated threonine while maintaining a negative charge. Carbodiimide (R'-N-C-N-R') (including, for example, 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-nitro cationic-4,4-dimethylpentyl) carbodiimide) to selectively modify carboxyl side groups (for example, asparaginyl or glutamyl). Can also be reacted with ammonium ion Asparagyl or glutamyl groups are converted to asparagyl and glutamyl groups.

可藉由(例如)除離胺酸及精胺酸以外利用胺基酸鳥胺酸、瓜胺酸或(胍基)-乙酸或(胍基)烷基-乙酸(其中烷基可經甲基、乙基、丙基、己基、丁基、戊基、異丙基、異丁基、第二異丁基、異戊基取代或未經取代)或非酸性胺基酸取代來生成鹼性胺基酸之模擬物。可用腈衍生物(例如，含有CN-部分替代COOH)取代天冬醯胺或麩醯胺酸。可使天冬醯胺基及麩醯胺基殘基脫氮基成為相應的天冬胺醯基或麩胺醯基殘基。 This can be achieved by, for example, utilizing the amino acids ornithine, citrulline, or (guanidino)-acetic acid or (guanidino)alkyl-acetic acid (where the alkyl group can be replaced by a methyl group) in addition to lysine and arginine. , ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, second isobutyl, isopentyl substituted or unsubstituted) or non-acidic amino acid substitution to generate basic amines Amino acid mimics. Nitrile derivatives (eg, containing CN-moieties in place of COOH) can be used in place of asparagine or glutamic acid. Asparaginyl and glutamyl residues can be denitrogenated to the corresponding asparaginyl or glutamyl residues.

可藉由使精胺醯基與一或多種試劑(包括(例如)苯基乙二醛、2,3-丁二酮、1,2-環己烷二酮或寧海準(ninhydrin))視情況在鹼性條件下反應來生成精胺酸模擬物。可藉由使酪胺醯基與芳香族重氮化合物或四硝基甲烷反應生成酪胺酸殘基模擬物。可分別使用N-乙醯基咪唑及四硝基甲烷形成O-乙醯基酪胺醯基物質及3-硝基衍生物。 Optionally, the spermidyl group can be reacted with one or more reagents including, for example, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin. React under alkaline conditions to generate arginine mimics. Tyrosine residue mimetics can be generated by reacting tyrosyl groups with aromatic diazonium compounds or tetranitromethane. N-acetylimidazole and tetranitromethane can be used to form O-acetyltyramide species and 3-nitro derivatives, respectively.

可藉由使離胺醯基與琥珀酸酐或其他羧酸酸酐反應生成(且可改變胺基末端殘基)離胺酸模擬物。亦可藉由與亞胺酸酯(例如甲基吡啶亞胺酸甲酯、磷酸吡哆醛、吡哆醛、氯硼氫化物、三硝基苯磺酸、O-甲基異脲、2,4-戊烷二酮)反應及轉醯胺基酶催化之與乙醛酸鹽之反應來生成離胺酸及其他含α胺基之殘基模擬物。 Lysine mimetics can be generated (and can alter the amino terminal residue) by reacting lysine groups with succinic anhydride or other carboxylic acid anhydrides. It can also be combined with imidate (such as methyl pyridine imidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2, 4-pentanedione) reaction and transamidase-catalyzed reaction with glyoxylate to generate lysine and other α-amine-containing residue mimics.

可藉由與甲硫胺酸亞碸反應生成甲硫胺酸模擬物。脯胺酸模擬物包括(例如)六氫吡啶羧酸、噻唑啶羧酸、3-羥基脯胺酸或4-羥基脯胺酸、去氫脯胺酸、3-甲基脯胺酸或4-甲基脯胺酸及3,3,-二甲基脯胺酸。可藉由使組胺醯基與二乙基焦碳酸酯或對溴苯甲醯甲基溴化物反應生成組胺酸模擬物。其他模擬物包括(例如)藉由以下生成之彼等：脯胺酸及離胺酸之羥基化；絲胺醯基或蘇胺醯基殘基之羥基之磷酸化；離胺酸、精胺酸及組胺酸之α-胺基之甲基化；N-末端胺之乙醯化；主鏈醯胺殘基之甲基化或利用N-甲基胺基酸取代；或C-末端羧基之醯胺化。 Methionine mimetics can be generated by reaction with methionine sulfone. Proline mimetics include, for example, hexahydropyridinecarboxylic acid, thiazolidinecarboxylic acid, 3-hydroxyproline or 4-hydroxyproline, dehydroproline, 3-methylproline or 4- Methylproline and 3,3,-dimethylproline. histamine Acyl groups react with diethylpyrocarbonate or p-bromobenzoylmethyl bromide to generate histidine mimetics. Other mimetics include, for example, those generated by: hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of serinyl or threonyl residues; lysine, arginine and the methylation of the α-amino group of histidine; the acetylation of the N-terminal amine; the methylation or substitution of the main chain amide residue with N-methyl amino acid; or the acetylation of the C-terminal carboxyl group amidation.

一或多個殘基亦可由具有相反手性之胺基酸(或擬肽殘基)替代。因此，L-構形(端視化學實體之結構，其亦可稱作R或S)中天然存在之任一胺基酸可經相同但具有相反手性之胺基酸或模擬物(其稱作D-胺基酸，但其可另外稱作R形式或S形式)替代。 One or more residues may also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any naturally occurring amino acid in the L-configuration (which may also be referred to as R or S depending on the structure of the chemical entity) can be replaced by an amino acid of the same but opposite chirality or a mimetic (which is referred to as as the D-amino acid, but which may otherwise be referred to as the R-form or the S-form) instead.

本發明肽及擬肽進一步包括本文所闡述序列之經修飾形式，條件係該經修飾形式保留未經修飾或參考肽或擬肽之功能之至少一部分。舉例而言，經修飾肽或擬肽將保留細胞增殖抑制或G2廢除活性之至少一部分，但相對於參考肽或擬肽其可具有增加或降低之細胞增殖抑制或G2廢除活性。 The peptides and peptidomimetics of the invention further include modified forms of the sequences set forth herein, provided that the modified form retains at least a portion of the function of the unmodified or reference peptide or peptidomimetic. For example, a modified peptide or peptidomimetic will retain at least a portion of the cell proliferation inhibitory or G2 abrogating activity, but it may have increased or decreased cell proliferation inhibitory or G2 abrogating activity relative to a reference peptide or peptidomimetic.

經修飾肽及擬肽之一或多個胺基酸殘基可經另一殘基取代，添加至序列中或自序列缺失。在一個實施例中，經修飾肽或擬肽具有一或多個胺基酸取代、添加或缺失(例如，1至3個、3至5個、5至10個或更多個)。在一態樣中，取代係利用側鏈佔據與參考胺基酸或模擬物(正經取代之胺基酸或模擬物)類似之空間之胺基酸或模擬物進行。在再一態樣中，取代係利用在結構上與人類殘基類似之非人類胺基酸進行。在具體態樣中，取代係保守胺基酸取代。 Modified peptides and peptidomimetics can have one or more amino acid residues substituted, added to, or deleted from the sequence by another residue. In one embodiment, a modified peptide or peptidomimetic has one or more amino acid substitutions, additions or deletions (eg, 1 to 3, 3 to 5, 5 to 10 or more). In one aspect, the substitution is made with an amino acid or mimetic whose side chain occupies a similar space as the reference amino acid or mimetic (the amino acid or mimetic being substituted). In yet another aspect, substitutions are made with non-human amino acids that are structurally similar to human residues. In specific aspects, the substitutions are conservative amino acid substitutions.

如本文所用術語「類似空間」意指佔據在大小方面與參考部分類似之三維空間之化學部分。通常，佔據類似空間之部分之大小將與參考部分類似。「佔據類似側鏈空間」之胺基酸或模擬物之側鏈所佔三維空間之大小與參考胺基酸或模擬物類似。d-(Phe-2,3,4,5,6-F)、l-(Phe-2,3,4,5,6-F)、d-(Phe-3,4,5F)、l-(Phe-3,4,5F)、d-(Phe-4CF3)或l-(Phe-4CF3)之特定實例係(l或d-Phe-2R1,3R2,4R3,5R4,6R5)，其中R1、R2、R3、R4、R5可為氯化物、溴化物、氟化物、碘化物、氫、氧化氫或不存在。對於小分子(例如具有約1埃(Angstrom)大小之氟化物)，類似空間可缺少一部分。 The term "similar space" as used herein means a chemical moiety occupying a three-dimensional space similar in size to a reference moiety. Typically, the size of the portion occupying a similar space will be similar to the reference portion. The size of the three-dimensional space occupied by the side chains of amino acids or mimics that "occupy similar side chain space" Similar to reference amino acid or mimetic. d-(Phe-2,3,4,5,6-F), l-(Phe-2,3,4,5,6-F), d-(Phe-3,4,5F), l- A specific example of (Phe-3,4,5F), d-(Phe-4CF3) or 1-(Phe-4CF3) is (1 or d-Phe-2R1,3R2,4R3,5R4,6R5), wherein R1, R2, R3, R4, R5 can be chloride, bromide, fluoride, iodide, hydrogen, hydrogen oxide or not present. For small molecules, such as fluoride with a size of about 1 Angstrom, a portion of the similar space may be missing.

術語「保守取代」意味著一個胺基酸經生物上、化學上或結構上類似之殘基替代。在生物上類似意味著取代與生物活性(例如抗細胞增殖或G2廢除活性)相容。在結構上類似意味著胺基酸具有帶有類似長度(例如丙胺酸、甘胺酸及絲胺酸)或具有類似大小之側鏈。化學類似性意味著殘基具有相同電荷或均具有親水性或疏水性。具體實例包括一個疏水殘基(例如異白胺酸、纈胺酸、白胺酸或甲硫胺酸)取代另一者，或一個極性殘基取代另一者，例如精胺酸取代離胺酸，麩胺酸取代天冬胺酸，或麩醯胺酸取代天冬醯胺，絲胺酸取代蘇胺酸，及諸如此類。 The term "conservative substitution" means that an amino acid is replaced by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution is compatible with biological activity, such as anti-cell proliferation or G2 abrogating activity. Structurally similar means that the amino acids have side chains of similar length (eg, alanine, glycine, and serine) or are of similar size. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Specific examples include substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another, or substitution of one polar residue for another such as arginine for lysine , glutamic acid for aspartic acid, or glutamic acid for asparagine, serine for threonine, and the like.

因此，本發明肽及擬肽包括具有與表1中所述肽及擬肽之序列不一致之序列的肽及擬肽。在一個實施例中，肽或擬肽之序列與表1中所闡述之序列具有50%、60%、70%、75%、80%、85%、90%、95%或更大一致性。在一態樣中，一致性係在序列之定義區域(例如胺基或羧基末端3至5個殘基)方面。 Thus, the peptides and peptidomimetics of the present invention include peptides and peptidomimetics having sequences that do not correspond to the sequences of the peptides and peptidomimetics described in Table 1. In one embodiment, the sequence of the peptide or peptidomimetic is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to the sequences set forth in Table 1. In one aspect, identity is over a defined region of the sequence (eg, the amine or carboxyl terminal 3 to 5 residues).

可使用業內已知之任一方法產生及分離本發明化合物(包括肽及擬肽)。可使用業內已知之化學方法完整或部分地合成肽(例如，參見Caruthers(1980)Nucleic Acids Res.Symp.Ser.215-223；Horn(1980)Nucleic Acids Res.Symp.Ser.225-232；及Banga,A.K.,Therapeutic Peptides and Proteins,Formulation,Processing and Delivery Systems(1995) Technomic Publishing Co.,Lancaster,PA)。可使用各種固相技術實施肽合成(例如，參見Roberge(1995)Science 269：202；Merrifield(1997)Methods Enzymol.289：3-13)且可使用(例如)ABI 431A Peptide Synthesizer(Perkin Elmer)根據製造商之說明書達成自動化合成。 Compounds of the invention, including peptides and peptidomimetics, can be produced and isolated using any method known in the art. Peptides can be synthesized in whole or in part using chemical methods known in the art (see, e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; and Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be performed using various solid-phase techniques (see, for example, Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and can be performed using, for example, the ABI 431A Peptide Synthesizer (Perkin Elmer) according to Automated synthesis was achieved according to the manufacturer's instructions.

可使用業內已知之各種程序及方法合成納入個別合成殘基及多肽之模擬物(例如，參見Organic Syntheses Collective Volumes,Gilman等人(編輯)John Wiley & Sons,Inc.,NY)。亦可使用組合方法合成肽及肽模擬物。生成肽及擬肽文庫之技術已為人熟知，且包括(例如)多針(multipin)、茶包(tea bag)及分開-結合-混合(split-couple-mix)技術(例如參見al-Obeidi(1998)Mol.Biotechnol.9：205-223；Hruby(1997)Curr.Opin.Chem.Biol.1：114-119；Ostergaard(1997)Mol.Divers.3：17-27；及Ostresh(1996)Methods Enzymol.267：220-234)。可藉由化學修飾方法進一步產生經修飾肽(例如參見，Belousov(1997)Nucleic Acids Res.25：3440-3444；Frenkel(1995)Free Radic.Biol.Med.19：373-380；及Blommers(1994)Biochemistry 33：7886-7896)。 Mimetics incorporating individual synthetic residues and polypeptides can be synthesized using various procedures and methods known in the art (see, eg, Organic Syntheses Collective Volumes, Gilman et al. (eds.) John Wiley & Sons, Inc., NY). Combinatorial approaches can also be used to synthesize peptides and peptidomimetics. Techniques for generating peptide and peptidomimetic libraries are well known and include, for example, multipin, tea bag, and split-couple-mix techniques (see, for example, al-Obeidi (1998) Mol. Biotechnol. 9: 205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1: 114-119; Ostergaard (1997) Mol. Divers. 3: 17-27; and Ostresh (1996) Methods Enzymol. 267:220-234). Modified peptides can be further produced by chemical modification methods (see, for example, Belousov (1997) Nucleic Acids Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; and Blommers (1994) ) Biochemistry 33:7886-7896).

肽亦可合成且可作為具有一或多個與其連接之其他結構域之融合蛋白表現，用於產生更具免疫原性之肽，更易於分離以重組方式合成之肽，或鑑別及分離抗體或表現抗體之B細胞。有利於檢測及純化之結構域包括(例如)容許在經固定金屬上純化之金屬螯合肽，例如多組胺酸束及組胺酸-色胺酸模組；容許在經固定免疫球蛋白上純化之蛋白質A結構域；及用於FLAGS擴散/親和純化系統(Immunex Corp,Seattle WA)中之結構域。可使用在純化結構域與肽之間納入可裂解連接體序列(例如Xa因子或腸激酶(Invitrogen,San Diego CA))來促進肽純化。舉例而言，表現載體可包括與 6個組胺酸殘基連接之編碼肽之核酸序列，隨後硫氧還蛋白及腸激酶裂解位點(例如，參見Williams(1995)Biochemistry 34：1787-1797；Dobeli(1998)Protein Expr.Purif.12：404-14)。組胺酸殘基有利於檢測及純化融合蛋白，同時腸激酶裂解位點提供自融合蛋白之其餘部分純化肽之方式。涉及編碼融合蛋白之載體及應用融合蛋白之技術在業內已知(例如，參見Kroll(1993)DNA Cell.Biol.,12：441-53)。 Peptides can also be synthesized and can be expressed as fusion proteins with one or more additional domains linked thereto for the generation of more immunogenic peptides, for easier isolation of recombinantly synthesized peptides, or for the identification and isolation of antibodies or Antibody-expressing B cells. Domains that facilitate detection and purification include, for example, metal-chelating peptides that allow purification on immobilized metals, such as polyhistidine tracts and histidine-tryptophan modules; Purified protein A domain; and domain used in the FLAGS diffusion/affinity purification system (Immunex Corp, Seattle WA). Peptide purification can be facilitated using the inclusion of a cleavable linker sequence such as Factor Xa or enterokinase (Invitrogen, San Diego CA) between the purification domain and the peptide. For example, presentation vectors can include A nucleic acid sequence encoding a peptide linked by six histidine residues, followed by a thioredoxin and enterokinase cleavage site (see, for example, Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-14). The histidine residue facilitates detection and purification of the fusion protein, while the enterokinase cleavage site provides a means of purifying the peptide from the rest of the fusion protein. Techniques involving vectors encoding fusion proteins and applying fusion proteins are known in the art (see, eg, Kroll (1993) DNA Cell. Biol., 12:441-53).

本發明進一步提供編碼本發明肽之核酸。在特定實施例中，核酸編碼具有約8至12個、12至15個、15至18個、15至20個、18至25個、20至25個、25至35個、25至50個或50至100個胺基酸之長度或長度更大之本發明肽序列。 The present invention further provides nucleic acids encoding the peptides of the present invention. In particular embodiments, the nucleic acid code has about 8 to 12, 12 to 15, 15 to 18, 15 to 20, 18 to 25, 20 to 25, 25 to 35, 25 to 50 or Peptide sequences of the invention of 50 to 100 amino acids in length or greater.

術語「核酸」及「多核苷酸」在本文可互換使用，其係指核酸之所有形式，包括去氧核糖核酸(DNA)及核糖核酸(RNA)。核酸可為雙鏈的、單鏈的或三螺旋的、線性的或環狀的。核酸包括基因體DNA、cDNA及反義DNA 。RNA核酸可為經剪接或未剪接之mRNA、rRNA、tRNA或反義RNA(例如，RNAi)。本發明之核酸包括天然核酸、合成核酸以及核苷酸類似物及衍生物。該等經改變或經修飾多核苷酸包括提供(例如)核酸酶抗性之類似物。核酸長度亦可小於所例示之肽序列。舉例而言，肽序列中之任一者之子序列可編碼具有抗增殖或G2廢除活性之肽。 The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). A nucleic acid can be double-stranded, single-stranded or triple-helical, linear or circular. Nucleic acid includes genomic DNA, cDNA and antisense DNA. An RNA nucleic acid can be spliced or unspliced mRNA, rRNA, tRNA, or antisense RNA (eg, RNAi). Nucleic acids of the present invention include natural nucleic acids, synthetic nucleic acids, and nucleotide analogs and derivatives. Such altered or modified polynucleotides include analogs that provide, for example, nuclease resistance. Nucleic acid lengths can also be smaller than the exemplified peptide sequences. For example, a subsequence of any of the peptide sequences may encode a peptide with antiproliferative or G2 abrogating activity.

核酸可使用各種熟知之標準選殖及化學合成方法中之任一者來產生且可藉由熟習此項技術者已知之定點誘變或其他重組體技術進行故意改變。可藉助測序、凝膠電泳及諸如此類測定多核苷酸之純度。 Nucleic acids can be produced using any of a variety of well-known standard methods of cloning and chemical synthesis and can be deliberately altered by site-directed mutagenesis or other recombinant techniques known to those skilled in the art. The purity of polynucleotides can be determined by sequencing, gel electrophoresis, and the like.

可將本發明之核酸插入其中核酸之表現可受「表現控制元件」(稱作「表現盒」之組合)影響或調控之核酸構築體中。術語「表現控制元件」意指調控或影響與其可操作連接之核酸序列之表現之一或多個序列元件。可操作連接至核酸序列之表現控制元件控制轉錄及(若適當)核酸序列之轉譯。 A nucleic acid of the invention can be inserted into a nucleic acid construct in which the expression of the nucleic acid can be influenced or regulated by "expression control elements" (combinations referred to as "expression cassettes"). The term "presentation control element" means Refers to one or more sequence elements that regulate or affect the expression of a nucleic acid sequence to which it is operably linked. Expression control elements operably linked to a nucleic acid sequence control the transcription and, if appropriate, translation of the nucleic acid sequence.

術語「可操作連接」係指其中如此闡述之組份之關係允許該等組份以其預期方式起作用之功能並列。通常，表現控制元件在基因之5’端或3’端並列但亦可為內含子。啟動子通常位於編碼序列之5’。「啟動子」意指足以引導轉錄之最小序列元件。 The term "operably linked" refers to a functional juxtaposition of components so stated in a relationship which permits those components to function in their intended manner. Typically, expression control elements are juxtaposed at the 5' or 3' end of the gene but may also be introns. A promoter is usually located 5' to the coding sequence. "Promoter" means the smallest sequence element sufficient to direct transcription.

表現控制元件在蛋白質編碼基因之前方包括啟動子、增強子、轉錄終止子、基因沉默子、起始密碼子(例如，ATG)。表現控制元件活化組成型轉錄、可誘導轉錄(即，需要外部信號用於活化)或抑制轉錄(即，信號關閉轉錄；去除信號可活化轉錄)。表現盒亦可包括足以使基因表現對於特定細胞類型或組織可控制之控制元件(即，組織特異性控制元件)。 Expression control elements include promoters, enhancers, transcription terminators, gene silencers, initiation codons (eg, ATG) in front of protein-coding genes. Expression control elements activate constitutive transcription, inducible transcription (ie, require an external signal for activation), or repress transcription (ie, signal turns transcription off; removal of the signal activates transcription). The expression cassette may also include control elements sufficient to render gene expression controllable for a particular cell type or tissue (ie, tissue-specific control elements).

可將本發明之核酸插入質體中用於繁殖至宿主細胞中及用於後續遺傳操縱。質體係可在宿主細胞中穩定繁殖之核酸；質體視情況含有表現控制元件以便驅動宿主細胞中核酸編碼肽之表現。術語「載體」在本文中與質體同義使用且亦可包括用於在宿主細胞中表現之表現控制元件。質體及載體通常至少含有用於在細胞中繁殖之複製起點及啟動子。質體及載體因此可用於遺傳操縱肽編碼核酸、產生肽及在(例如)宿主細胞或整個生物體中表現肽。 The nucleic acids of the invention can be inserted into plastids for propagation into host cells and for subsequent genetic manipulation. Plastid A nucleic acid capable of stably propagating in a host cell; the plastid optionally contains expression control elements to drive expression of the nucleic acid-encoded peptide in the host cell. The term "vector" is used herein synonymously with plastid and may also include expression control elements for expression in a host cell. Plastids and vectors generally contain at least an origin of replication and a promoter for propagation in cells. Plastids and vectors are thus useful for genetic manipulation of peptide-encoding nucleic acids, production of peptides, and expression of peptides, eg, in host cells or whole organisms.

因此，肽可在以下各項中表現：使用組成型啟動子(例如T7)或可誘導啟動子(例如噬菌體□之pL、plac、ptrp、ptac(ptrp-lac雜交啟動子))之細菌系統；使用組成型啟動子(例如ADH或LEU2)或可誘導啟動子(例如GAL)之酵母系統(例如，參見Ausubel等人編輯之Current Protocols in Molecular Biology，第2卷，第13章，Greene Publish.Assoc.& Wiley Interscience,1988；Grant等人，Methods in Enzymology,153：516(1987),Wu及Grossman編輯；Bitter Methods in Enzymology,152：673(1987),Berger及Kimmel編輯，Acad.Press,N.Y.；及Strathern等人，The Molecular Biology of the Yeast Saccharomyces(1982)編輯Cold Spring Harbor Press，第I卷及第II卷；R.Rothstein之DNA Cloning,A Practical Approach，第11卷，第3章，D.M.Glover編輯，IRL Press,Wash.,D.C.,1986)；使用組成型或可誘導啟動子(例如蛻皮激素)之昆蟲細胞系統；及使用組成型啟動子(例如SV40、RSV)或源於哺乳動物細胞之基因體(例如金屬硫蛋白IIA啟動子、熱激啟動子)或源於哺乳動物病毒(例如腺病毒晚期啟動子)或可誘導小鼠乳房腫瘤病毒長末端重複之可誘導啟動子之哺乳動物細胞系統。肽表現系統進一步包括經設計用於活體內使用之載體，包括腺病毒載體(美國專利第5,700,470號及第5,731,172號)、腺相關載體(美國專利第5,604,090號)、單純皰疹病毒載體(美國專利第5,501,979號)及反轉錄病毒載體(美國專利第5,624,820號、第5,693,508號及第5,674,703號及WIPO公開案WO92/05266及WO92/14829)。基因療法中亦已採用牛乳頭瘤病毒(BPV)(美國專利第5,719,054號)。該等基因療法載體亦包括基於CMV之載體(美國專利第5,561,063號)。 Thus, peptides can be expressed in bacterial systems using constitutive promoters (such as T7) or inducible promoters (such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of bacteriophage D); Yeast systems using constitutive promoters (such as ADH or LEU2) or inducible promoters (such as GAL) (see, for example, Current Protocols in Molecular Biology edited by Ausubel et al., Vol. 2, Chapter 13, Greene Publish. Assoc .& Wiley Interscience, 1988; Grant et al., Methods in Enzymology , 153:516 (1987), edited by Wu and Grossman; Bitter Methods in Enzymology , 152:673 (1987), edited by Berger and Kimmel, Acad.Press, NY; and Strathern et al., The Molecular Biology of the Yeast Saccharomyces (1982) ed. Cold Spring Harbor Press, Vol. I and II; DNA Cloning, A Practical Approach by R. Rothstein, Vol. 11, Chapter 3, edited by DMGlover , IRL Press, Wash., DC, 1986); insect cell systems using constitutive or inducible promoters (e.g. ecdysone); and genes using constitutive promoters (e.g. SV40, RSV) or derived from mammalian cells mammalian cell systems derived from mammalian viruses (e.g. adenovirus late promoter) or inducible promoters that induce mouse mammary tumor virus long terminal repeats . Peptide expression systems further include vectors designed for in vivo use, including adenoviral vectors (US Pat. Nos. 5,700,470 and 5,731,172), adeno-associated vectors (US Pat. 5,501,979) and retroviral vectors (US Patent Nos. 5,624,820, 5,693,508 and 5,674,703 and WIPO Publications WO 92/05266 and WO 92/14829). Bovine papilloma virus (BPV) has also been employed in gene therapy (US Patent No. 5,719,054). Such gene therapy vectors also include CMV-based vectors (US Patent No. 5,561,063).

因此，本發明亦提供插入宿主細胞中編碼本發明肽之核酸。在一個實施例中，宿主細胞係原核細胞。在另一實施例中，宿主細胞係真核細胞。在各個態樣中，真核細胞係酵母或哺乳動物(例如，人類、靈長類動物等)細胞。 Accordingly, the present invention also provides for insertion into a host cell of a nucleic acid encoding a peptide of the present invention. In one embodiment, the host cell is a prokaryotic cell. In another embodiment, the host cell is a eukaryotic cell. In various aspects, eukaryotic cells are yeast or mammalian (eg, human, primate, etc.) cells.

如本文所用，「宿主細胞」係引入可繁殖、轉錄或編碼所表現肽之核酸之細胞。該術語亦包括個體宿主細胞之任一後代。 As used herein, a "host cell" introduces a nucleus capable of propagating, transcribing, or encoding the expressed peptide. acid cells. The term also includes any descendants of an individual host cell.

宿主細胞包括(但不限於)微生物(例如細菌或酵母)；及植物、昆蟲及哺乳動物細胞。舉例而言，本發明提供經重組體噬菌體核酸、質體核酸或黏粒核酸表現載體轉化之細菌；經重組體酵母表現載體轉化之酵母；經重組體病毒表現載體(例如，花椰菜嵌紋病毒CaMV；菸草嵌紋病毒TMV)感染或經重組體質體表現載體(例如，Ti質體)轉化之植物細胞系統；經重組體病毒表現載體(例如，桿狀病毒)感染之昆蟲細胞系統；或經重組體病毒表現載體(例如，反轉錄病毒、腺病毒、牛痘病毒)感染之動物細胞系統，或經改造用於穩定表現之經轉化動物細胞系統。 Host cells include, but are not limited to, microorganisms such as bacteria or yeast; and plant, insect, and mammalian cells. For example, the present invention provides bacteria transformed with recombinant phage nucleic acid, plastid nucleic acid or cosmid nucleic acid expression vector; yeast transformed with recombinant yeast expression vector; recombinant virus expression vector (for example, cauliflower mosaic virus CaMV ; Tobacco Mosaic Virus TMV) infection or plant cell system transformed by recombinant plastid expression vector (for example, Ti plastid); insect cell system infected by recombinant virus expression vector (for example, baculovirus); or by recombinant Animal cell systems infected with somatic viral expression vectors (eg, retroviruses, adenoviruses, vaccinia viruses), or transformed animal cell systems engineered for stable expression.

表現載體亦可含有編碼賦予抗選擇性壓力或可鑑別標記物(例如，□-半乳糖苷酶)之抗性之可選標記物之核酸，藉此容許具有載體之細胞鑑別、生長及擴增。或者，可選標記物可在第二載體上，該第二載體與含有本發明多核苷酸之第一載體共轉染至宿主細胞中。可使用眾多選擇系統，包括(但不限於)單純皰疹病毒胸苷激酶基因(Wigler等人，Cell 11：223(1977))、次黃嘌呤-鳥嘌呤磷酸核糖基轉移酶基因(Szybalska等人，Proc.Natl.Acad.Sci.USA 48：2026(1962))，且腺嘌呤磷酸核糖基轉移酶(Lowy等人，Cell 22：817(1980))基因，其可分別用於tk細胞、hgprt細胞或aprt細胞中。可使用抗代謝物抗性作為針對以下基因進行選擇之基礎：dhfr，其賦予抗胺甲喋呤之抗性(O'Hare等人，Proc.Natl.Acad.Sci.USA 78：1527(1981))；apt基因，其賦予抗黴酚酸(mycophenolic acid)之抗性(Mulligan等人，Proc.Natl.Acad.Sci.USA 78：2072(1981))；新黴素(neomycin)基因，其賦予抗胺基醣苷G-418之抗性(Colberre-Garapin等人，J.Mol.Biol.150：1(1981))；及潮黴素(hygromycin)基因，其賦予抗潮黴素之抗性 (Santerre等人，Gene 30：147(1984))。其他可選擇基因包括trpB，其容許細胞利用吲哚代替色胺酸；hisD，其容許細胞利用組胺醇代替組胺酸(Hartman等人，Proc.Natl.Acad.Sci.USA 85：8047(1988))；及ODC(鳥胺酸去羧酶)，其賦予對鳥胺酸去羧酶抑制劑2-(二氟甲基)-DL-鳥胺酸DFMO之抗性(McConlogue(1987)：Current Communications in Molecular Biology,Cold Spring Harbor Laboratory)。 Expression vectors may also contain nucleic acid encoding a selectable marker that confers resistance to selective pressure or an identifiable marker (e.g., D-galactosidase), thereby allowing identification, growth, and expansion of cells bearing the vector . Alternatively, the selectable marker can be on a second vector that is co-transfected into the host cell with the first vector containing the polynucleotide of the invention. A number of selection systems are available including, but not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al., Cell 11:223 (1977)), the hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al. , Proc.Natl.Acad.Sci.USA 48:2026 (1962)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) gene, which can be used in tk cells, hgprt cells or aprt cells. Antimetabolite resistance can be used as the basis for selection against the gene dhfr , which confers resistance to methotrexate (O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981) ); the apt gene, which confers resistance to mycophenolic acid (Mulligan et al., Proc.Natl.Acad.Sci.USA 78:2072 (1981)); the neomycin ( neomycin ) gene, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981)); and the hygromycin gene, which confers resistance to hygromycin ( Santerre et al., Gene 30:147 (1984)). Other selectable genes include trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to use histidinol instead of histidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047 (1988 )); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2-(difluoromethyl)-DL-ornithine DFMO (McConlogue (1987): Current Communications in Molecular Biology , Cold Spring Harbor Laboratory).

如本文所用，術語「核酸損害治療」及「核酸損害劑」意指直接或間接損害核酸(例如，DNA、cDNA、基因體DNA、mRNA、tRNA或rRNA)之任一治療方案。該等藥劑之特定實例包括烷基化劑、亞硝基脲、抗代謝物、植物鹼、植物提取物及放射性同位素。藥劑之特定實例亦包括核酸損害性藥物，例如，5-氟尿嘧啶(5-FU)、卡培他濱(capecitabine)、S-1(替加氟(Tegafur)、5-氯-2,4-二羥基吡啶及氧嗪酸(oxonic acid))、5-乙炔基尿嘧啶、阿拉伯糖基胞嘧啶(阿糖胞苷)、5-氮胞苷(5-AC)、2’,2’-二氟-2’-去氧胞苷(dFdC)、嘌呤抗代謝物(巰嘌呤、硫唑嘌呤、硫鳥嘌呤)、鹽酸吉西他濱(gemcitabine hydrochloride)(健擇)、噴司他汀(pentostatin)、別嘌呤醇、2-氟-阿拉伯糖基-腺嘌呤(2F-ara-A)、羥基脲、硫芥(雙氯乙基二硫化物)、甲基二(氯乙基)胺、美法侖、氮芥苯丁酸、環磷醯胺、異環磷醯胺、噻替派(thiotepa)、AZQ、絲裂黴素C、環氧乳醇、二溴衛矛醇、磺酸烷基酯(白消安)、亞硝基脲(BCNU,CCNU,4-甲基CCNU或ACNU)、丙卡巴肼、達卡巴嗪(decarbazine)、蝴蝶黴素、蒽環抗生素(例如多柔比星(阿德力黴素；ADR)、道諾黴素(西如比星(Cerubicine))、伊達比星(idarubicin)(去甲氧柔紅黴素(Idamycin))及泛艾黴素(epirubicin)(艾倫斯(Ellence)))、蒽環抗生素類似物(例如米托蒽醌(mitoxantrone))、放線菌素D、非插層拓樸異構酶抑制劑(例如表鬼臼毒素(依託泊苷(etoposide)=VP16、替尼泊苷(teniposide)=VM-26))、足葉草黴素(podophylotoxin)、博來黴素(Bleo)、派來黴素、與核酸形成加合物之化合物(包括鉑衍生物(例如，順鉑(CDDP)、順鉑之反式類似物、卡鉑、異丙鉑(iproplatin)、四鉑(tetraplatin)及奧沙利鉑))、喜樹鹼、托泊替康(topotecan)、伊立替康(irinotecan)(CPT-11)及SN-38。核酸損害治療之特定實例包括輻射(例如，紫外(UV)輻射、紅外(IR)輻射或α輻射、β輻射或γ-輻射)及環境衝擊(例如，發熱)。 As used herein, the terms "nucleic acid damaging treatment" and "nucleic acid damaging agent" mean any treatment regimen that directly or indirectly damages nucleic acid (eg, DNA, cDNA, genomic DNA, mRNA, tRNA or rRNA). Specific examples of such agents include alkylating agents, nitrosoureas, antimetabolites, plant alkaloids, plant extracts, and radioisotopes. Specific examples of agents also include nucleic acid damaging drugs, for example, 5-fluorouracil (5-FU), capecitabine, S-1 (Tegafur, 5-chloro-2,4-di Hydroxypyridine and oxonic acid (oxonic acid)), 5-ethynyluracil, arabinosylcytosine (cytarabine), 5-azacytidine (5-AC), 2',2'-difluoro -2'-Deoxycytidine (dFdC), purine antimetabolites (mercaptopurine, azathioprine, thioguanine), gemcitabine hydrochloride (Gemzeta), pentostatin, allopurinol , 2-fluoro-arabinosyl-adenine (2F-ara-A), hydroxyurea, sulfur mustard (dichloroethyl disulfide), methyl di(chloroethyl)amine, melphalan, nitrogen mustard Phenylbutyric acid, cyclophosphamide, ifosfamide, thiotepa, AZQ, mitomycin C, epoxylactol, dibromodulcitol, alkyl sulfonate (busulfan ), nitrosoureas (BCNU, CCNU, 4-methyl CCNU or ACNU), procarbazine, decarbazine, tritemycin, anthracyclines (such as doxorubicin (adromycin ; ADR), daunorubicin (Cerubicine), idarubicin (Idamycin), and epirubicin (Ellence ))), anthracycline analogs (eg mitoxantrone), actinomycin D, non-intercalating topoisomers Enzyme inhibitors (e.g. epipodophyllotoxin (etoposide = VP16, teniposide = VM-26)), podophylotoxin, bleomycin (Bleo), Pelemycin, compounds that form adducts with nucleic acids (including platinum derivatives such as cisplatin (CDDP), trans analogs of cisplatin, carboplatin, iproplatin, tetraplatin and oxaliplatin)), camptothecin, topotecan, irinotecan (CPT-11) and SN-38. Specific examples of nucleic acid damaging treatments include radiation (eg, ultraviolet (UV) radiation, infrared (IR) radiation, or alpha, beta, or gamma-radiation) and environmental shock (eg, fever).

如本文所用，術語「抗增殖治療」及「抗增殖劑」意指無論該治療或藥劑是否損害核酸均直接或間接抑制細胞、病毒、細菌或其他單細胞性或多細胞性生物體之增殖之任一治療方案。抗增殖劑之具體實例係抗腫瘤及抗病毒藥物，其抑制細胞增殖或病毒增殖或複製。特定實例尤其包括環磷醯胺、硫唑嘌呤、環孢素A、普賴蘇濃、美法侖、氮芥苯丁酸、甲基二(氯乙基)胺、白消安、胺甲喋呤、6-巰嘌呤、硫鳥嘌呤、胞嘧啶阿拉伯糖苷、紫杉醇、長春鹼、長春新鹼、多柔比星、放線菌素D、光輝黴素、卡莫司汀、洛莫司汀、司莫司汀、鏈佐黴素、羥基脲、順鉑、米托坦、丙卡巴肼、達卡巴嗪及二溴甘露醇。造成核酸複製錯誤或抑制核酸複製之抗增殖劑，例如核苷及核苷酸類似物(例如，AZT或5-AZC)。 As used herein, the terms "antiproliferative therapy" and "antiproliferative agent" mean any agent that directly or indirectly inhibits the proliferation of cells, viruses, bacteria, or other unicellular or multicellular organisms whether or not the treatment or agent damages nucleic acid. any treatment option. Specific examples of antiproliferative agents are antineoplastic and antiviral drugs, which inhibit cell proliferation or viral proliferation or replication. Specific examples include, inter alia, cyclophosphamide, azathioprine, cyclosporine A, presulon, melphalan, mechlorethamine, methyldi(chloroethyl)amine, busulfan, methotrene Glycine, 6-mercaptopurine, thioguanine, cytosine arabinoside, paclitaxel, vinblastine, vincristine, doxorubicin, actinomycin D, luminomycin, carmustine, lomustine, Mustin, streptozotocin, hydroxyurea, cisplatin, mitotane, procarbazine, dacarbazine, and dibromomannitol. Antiproliferative agents that cause nucleic acid replication errors or inhibit nucleic acid replication, such as nucleosides and nucleotide analogs (eg, AZT or 5-AZC).

本發明肽及擬肽亦可加強微管穩定劑或去穩定劑(例如長春花生物鹼(長春鹼=VLB、長春新鹼=VCR、長春瑞濱(vinorelbine)=VRLB、長春氟寧(vinflunine)=VFL)及紫杉烷(太平洋紫杉醇(paclitaxel)及多西他賽(docetaxel)=泰索帝(taxotare)))之抗細胞增殖活性。因此，該等藥劑可進一步包括在本發明之組合物中並用於本發明之方法中。 The peptides and peptidomimetics of the present invention can also potentiate microtubule stabilizers or destabilizers (such as vinca alkaloids (vinblastine=VLB, vincristine=VCR, vinorelbine=VRLB, vinflunine) = VFL) and the antiproliferative activity of taxanes (paclitaxel and docetaxel = taxotare). Accordingly, such agents may further be included in the compositions of the invention and used in the methods of the invention.

可利用本發明化合物處理之細胞包括期望在活體外、離體或活體內抑制或預防其增殖之任一細胞。具體靶細胞展現短於正常細胞週期之G1查核點時間或具有受損之細胞週期G1查核點，使得細胞在經過足夠時間以完成核酸修復之前離開G1查核點。因此，候選者細胞包括無論細胞係正常的抑或異常的均快速增殖之細胞。特定實例係良性細胞或腫瘤性細胞、轉移細胞或非轉移細胞。其他候選者細胞可藉由量測其增殖速率或細胞留在G1期之時間長度來鑑別。候選者細胞亦可藉由使測試細胞與單獨的本發明化合物或其與核酸損害治療之組合接觸及確定經接觸細胞是否展現降低之增殖或增加之細胞死亡或細胞凋亡/劇變來鑑別。 Cells that may be treated with the compounds of the invention include cells that are desired to be inhibited in vitro, ex vivo or in vivo. Any cell that inhibits or prevents its proliferation. A particular target cell exhibits a shorter than normal cell cycle G1 checkpoint time or has an impaired cell cycle G1 checkpoint such that the cell exits the G1 checkpoint before sufficient time has elapsed to complete nucleic acid repair. Thus, candidate cells include rapidly proliferating cells, whether normal or abnormal of cell lineage. Particular examples are benign or neoplastic cells, metastatic or non-metastatic cells. Other candidate cells can be identified by measuring their rate of proliferation or the length of time the cells remain in G1 phase. Candidate cells can also be identified by contacting test cells with a compound of the invention alone or in combination with a nucleic acid damaging treatment and determining whether the contacted cells exhibit reduced proliferation or increased cell death or apoptosis/catastrophe.

因此，本發明化合物及T細胞活化劑及/或免疫查核點抑制劑可用於抑制活體外、離體及活體內細胞增殖。因此，患有特徵在於細胞增殖或細胞存活異常或不合意或不期望或者細胞分化異常或有缺陷之病症或生理病況或具有患該病症或生理病況之風險的個體可利用單獨的本發明化合物或其與T細胞活化劑及/或免疫查核點抑制劑之組合來治療。 Accordingly, compounds of the invention and T cell activators and/or immune checkpoint inhibitors are useful for inhibiting cell proliferation in vitro, ex vivo and in vivo. Thus, individuals suffering from or at risk of developing a disorder or physiological condition characterized by abnormal or undesirable or undesirable cell proliferation or cell survival or abnormal or defective cell differentiation may utilize the compounds of the present invention alone or It is treated in combination with T cell activators and/or immune checkpoint inhibitors.

因此，根據本發明，提供抑制細胞增殖之方法、增加細胞對核酸損害劑或治療之敏感性之方法及增加對活體外、離體及活體內細胞之核酸損害之方法。在一個實施例中，方法包括使細胞(例如，經培養細胞或存於個體中之細胞)與足以抑制細胞增殖之量的本發明肽或擬肽及T細胞活化劑接觸。在另一實施例中，方法包括使細胞(例如，經培養細胞或存於個體中之細胞)與足以抑制細胞增殖之量之本發明肽或擬肽及免疫查核點抑制劑接觸。在另一實施例中，方法包括使細胞(例如，經培養細胞或存於個體中之細胞)與足以抑制細胞增殖之量之本發明肽或擬肽及T細胞活化劑及免疫查核點抑制劑接觸。 Thus, according to the present invention, there are provided methods of inhibiting cell proliferation, methods of increasing the sensitivity of cells to nucleic acid damaging agents or treatments, and methods of increasing nucleic acid damage to cells in vitro, ex vivo and in vivo. In one embodiment, the method comprises contacting a cell (eg, a cultured cell or a cell in an individual) with a peptide or peptidomimetic of the invention and a T cell activator in an amount sufficient to inhibit proliferation of the cell. In another embodiment, the method comprises contacting a cell (eg, a cultured cell or a cell in an individual) with a peptide or peptidomimetic and an immune checkpoint inhibitor of the invention in an amount sufficient to inhibit proliferation of the cell. In another embodiment, the method comprises administering to cells (e.g., cells in culture or in an individual) a peptide or peptidomimetic of the invention and a T cell activator and immune checkpoint inhibitor in an amount sufficient to inhibit cell proliferation. touch.

在另一實施例中，方法包括使細胞與足以增加細胞對核酸損害劑或治療之敏感性之量之本發明肽或擬肽及/或T細胞活化劑及/或免疫查核點抑制劑接觸。在另一實施例中，方法包括使細胞與足以增加細胞之核酸損害之量之本發明肽或擬肽及/或T細胞活化劑及/或免疫查核點抑制劑接觸。在各個態樣中，方法進一步包括使細胞與核酸損害劑接觸或使細胞暴露於核酸損害治療。 In another embodiment, the method comprises contacting the cell with an agent or therapeutic agent sufficient to increase the cell's resistance to nucleic acid damage. A therapeutically sensitive amount of a peptide or peptidomimetic of the invention and/or a T cell activator and/or an immune checkpoint inhibitor is contacted. In another embodiment, a method comprises contacting a cell with a peptide or peptidomimetic of the invention and/or a T cell activator and/or an immune checkpoint inhibitor in an amount sufficient to increase nucleic acid damage of the cell. In various aspects, the method further comprises contacting the cell with a nucleic acid damaging agent or exposing the cell to a nucleic acid damaging treatment.

本發明進一步提供治療個體之細胞增殖病症或分化病症之方法，該增殖病症或分化病症包括特徵在於細胞增殖或細胞存活不合意或不期望之病況、特徵在於細胞凋亡有缺陷或異常之病況、特徵在於細胞存活異常或有缺陷之病況以及特徵在於細胞分化異常或不充分之病況。在一個實施例中，方法包括向患有細胞增殖病症或具有患細胞增殖病症之風險之個體投與可有效治療細胞增殖病症之量之本發明肽或擬肽及/或T細胞活化劑及/或免疫查核點抑制劑。在一態樣中，該量足以改良個體之病況。在具體態樣中，改良包括至少一部分靶細胞(例如，異常增殖之細胞)改良、細胞增殖減少、細胞數量減少、抑制細胞數量增加、細胞凋亡增加或存活減少。在又一態樣中，在投與抑制細胞增殖之治療之前、與此同時或在此之後將本發明化合物投與個體。在其他具體態樣中，細胞增殖病症之細胞之至少一部分位於血液、乳房、肺、甲狀腺、頭或頸、腦、淋巴、胃腸道、生殖泌尿道、腎、胰臟、肝、骨、肌肉或皮膚中。 The invention further provides methods of treating a cell proliferative or differentiation disorder in an individual, including conditions characterized by undesirable or undesirable cell proliferation or cell survival, conditions characterized by defective or abnormal apoptosis, Conditions characterized by abnormal or defective cell survival and conditions characterized by abnormal or insufficient differentiation of cells. In one embodiment, the method comprises administering to an individual having or at risk of developing a cell proliferative disorder, a peptide or peptidomimetic and/or a T cell activator of the invention in an amount effective to treat the cell proliferative disorder and/or or immune checkpoint inhibitors. In one aspect, the amount is sufficient to ameliorate the condition in the subject. In specific aspects, modification includes modification of at least a portion of target cells (eg, abnormally proliferating cells), decreased cell proliferation, decreased cell number, increased suppressor cell number, increased apoptosis, or decreased survival. In yet another aspect, the compound of the invention is administered to the subject prior to, concurrently with, or after administration of a therapy that inhibits cellular proliferation. In other embodiments, at least a portion of the cells of the cell proliferative disorder are located in the blood, breast, lung, thyroid, head or neck, brain, lymph, gastrointestinal tract, genitourinary tract, kidney, pancreas, liver, bone, muscle, or in the skin.

在另一實施例中，方法包括向個體投與一定量之化合物及/或T細胞活化劑及/或免疫查核點抑制劑以治療實體腫瘤。在另一實施例中，方法包括向個體投與一定量之化合物及/或T細胞活化劑及/或免疫查核點抑制劑以治療液體腫瘤。在各個態樣中，在T細胞活化劑或免疫查核點抑制劑或另一抗腫瘤療法之前、與此同時或在此之後利用本發明化合物投與具有腫瘤之個體。 In another embodiment, the method comprises administering to an individual an amount of a compound and/or a T cell activator and/or an immune checkpoint inhibitor to treat a solid tumor. In another embodiment, the method comprises administering to an individual an amount of a compound and/or a T cell activator and/or an immune checkpoint inhibitor to treat a liquid tumor. In various aspects, administration of the compounds of the invention to patients with tumor the individual.

如本文所用，術語「增殖病症」及「增殖病況」意指特徵在於(例如，細胞、病毒、細菌、真菌等)增殖異常或不合意之任一病理學或非病理學生理病況。術語「細胞增殖病症」及「細胞增殖病況」意指特徵在於細胞增殖異常或不合意之任一病理學或非病理學生理病況，以及包括特徵在於細胞增殖或細胞存活不合意或不期望(例如，由細胞凋亡有缺陷引起)之病況、特徵在於細胞凋亡有缺陷或異常或有缺陷之病況以及特徵在於細胞存活異常或不合意或不期望之病況。術語「分化病症」意指特徵在於分化異常或有缺陷之任一病理學或非病理學生理病況。 As used herein, the terms "proliferative disorder" and "proliferative condition" mean any pathological or non-pathological physiological condition characterized by abnormal or undesirable proliferation (eg, of cells, viruses, bacteria, fungi, etc.). The terms "cell proliferative disorder" and "cell proliferative condition" mean any pathological or non-pathological physiological condition characterized by abnormal or undesired proliferation of cells, and includes those characterized by undesired or undesirable cell proliferation or cell survival (e.g. , conditions caused by defective apoptosis), conditions characterized by defective or abnormal or defective apoptosis, and conditions characterized by abnormal or undesirable or undesirable cell survival. The term "differentiation disorder" means any pathological or non-pathological physiological condition characterized by abnormal or defective differentiation.

適於治療之增殖或分化病症包括特徵在於細胞數量、細胞生長或細胞存活異常或不合意之疾病及非病理學生理病況(良性及贅瘤性)。因此，該等病症或病況可構成疾病狀態且包括所有類型之癌性生長或致癌過程、轉移性組織或惡性轉化之細胞、組織或器官，或可為非病理學的，亦即偏離正常但通常與疾病不相關。可根據本發明治療之非病理學病況之特定實例係來自創傷修復且引起結瘢之組織再生長。 Proliferative or differentiation disorders amenable to treatment include diseases and non-pathological physiological conditions (benign and neoplastic) characterized by abnormal or undesirable cell number, cell growth or cell survival. Accordingly, such disorders or conditions may constitute disease states and include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, or may be apathological, i.e. deviate from normal but usually Not associated with disease. A specific example of a non-pathological condition that may be treated according to the invention is tissue regrowth resulting from wound repair and causing scarring.

包含增殖或分化病症之細胞可聚集成細胞團塊或分散。術語「實體腫瘤」係指通常聚集在一起且形成團塊之贅瘤形成或轉移。具體實例包括內臟腫瘤，例如胃癌或結腸癌、肝細胞瘤、腎癌、肺及腦之腫瘤/癌症。「液體腫瘤」係指造血系統之贅瘤形成(例如淋巴瘤、骨髓瘤及白血病)或實際上擴散之贅瘤形成，因為其通常不形成實體團塊。白血病之具體實例包括急性及慢性淋巴母細胞性、成髓細胞性及多發性骨髓瘤。 Cells comprising a proliferation or differentiation disorder may aggregate into cell clumps or disperse. The term "solid tumor" refers to neoplastic formations or metastases that usually aggregate together and form clumps. Specific examples include visceral tumors such as stomach or colon cancer, hepatoma, kidney cancer, tumors/cancers of the lung and brain. "Liquid tumor" refers to neoplasms of the hematopoietic system (such as lymphomas, myelomas, and leukemias) or neoplasms that actually spread because they usually do not form solid masses. Specific examples of leukemia include acute and chronic lymphoblastic, myeloblastic and multiple myeloma.

該等病症包括實際上可影響任一細胞或組織類型之贅瘤或癌症，例如癌、肉瘤、黑色素瘤、轉移病症或造血性贅瘤性病症。轉移性腫瘤可源自多種原發性腫瘤類型，包括(但不限於)乳房、肺、甲狀腺、頭頸、腦、淋巴樣、胃腸(口腔、食管、胃、小腸、結腸、直腸)、生殖泌尿道(子宮、卵巢、子宮頸、膀胱、睪丸、陰莖、前列腺)、腎、胰臟、肝、骨、肌肉、皮膚等。 Such disorders include neoplasms or cancers that can affect virtually any cell or tissue type, such as carcinomas, sarcomas, melanomas, metastatic disorders, or hematopoietic neoplastic disorders. Metastatic tumors can originate from Multiple primary tumor types including (but not limited to) breast, lung, thyroid, head and neck, brain, lymphoid, gastrointestinal (mouth, esophagus, stomach, small intestine, colon, rectum), genitourinary (uterus, ovary, Cervix, bladder, testes, penis, prostate), kidney, pancreas, liver, bone, muscle, skin, etc.

癌係指上皮或內分泌組織之惡性病，且包括呼吸系統癌、胃腸系統癌、生殖泌尿系統癌、睪丸癌、乳房癌、前列腺癌、內分泌系統癌及黑色素瘤。實例性癌包括自子宮頸、肺、前列腺、乳房、頭頸、結腸、肝及卵巢形成之彼等。術語亦包括癌肉瘤，例如其包括由癌性及肉瘤性組織組成之惡性腫瘤。腺癌包括腺組織之癌，或其中腫瘤形成腺樣結構之癌。 Cancer refers to malignant diseases of epithelial or endocrine tissues, and includes respiratory system cancer, gastrointestinal system cancer, genitourinary system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. Exemplary cancers include those arising from the cervix, lung, prostate, breast, head and neck, colon, liver, and ovary. The term also includes carcinosarcoma, eg, which includes malignant tumors composed of cancerous and sarcomatous tissue. Adenocarcinoma includes carcinomas of glandular tissue, or carcinomas in which the tumor forms adenoid structures.

肉瘤係指間葉細胞起源性惡性腫瘤。實例性肉瘤包括(例如)淋巴肉瘤、脂肪肉瘤、骨肉瘤及纖維肉瘤。 Sarcomas are malignant tumors of mesenchymal origin. Exemplary sarcomas include, for example, lymphosarcoma, liposarcoma, osteosarcoma, and fibrosarcoma.

如本文所用術語「造血性增殖病症」意指涉及造血起源性增生/贅瘤細胞(例如由骨髓樣、淋巴樣或類紅血球譜系或其前體細胞引起)之疾病。通常，疾病由分化不良型急性白血病(例如成紅血球白血病及急性巨核細胞白血病)引起。其他實例性骨髓樣病症包括(但不限於)急性前骨髓性白血病(APML)、急性骨髓性白血病(AML)及慢性骨髓性白血病(CML)；淋巴樣惡性病包括(但不限於)急性淋巴母細胞性白血病(ALL)，其包括B譜系ALL及T譜系ALL、慢性淋巴球性白血病(CLL)、前淋巴球性白血病(PLL)、毛細胞白血病(HLL)及華氏巨球蛋白血症(Waldenstrom’s macroglobulinemia，WM)。其他惡性淋巴瘤包括(但不限於)非霍奇金氏淋巴瘤(non-Hodgkin lymphoma)及其變體、外周T細胞淋巴瘤、成人T細胞白血病/淋巴瘤(ATL)、皮膚T細胞淋巴瘤(CTCL)、大顆粒淋巴球性白血病(LGF)、霍奇金氏病及裡-斯病(Reed-Sternberg disease)。 The term "hematopoietic proliferative disorder" as used herein means a disease involving hyperplastic/neoplastic cells of hematopoietic origin, for example arising from cells of the myeloid, lymphoid or erythroid lineage or precursors thereof. Typically, the disease is caused by dysdifferentiated acute leukemias such as erythroblastic leukemia and acute megakaryoblastic leukemia. Other exemplary myeloid disorders include, but are not limited to, acute myeloid leukemia (APML), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML); lymphoid malignancies include, but are not limited to, acute lymphoid leukemia Cellular leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (Waldenstrom's Macroglobulinemia, WM). Other malignant lymphomas include (but are not limited to) non-Hodgkin lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

與本發明化合物組合使用之治療包括如本文所揭示或業內已知之任一抗增殖、核酸損害或抗腫瘤治療。舉例而言，抗細胞增殖或抗腫瘤治療可包含輻射治療或手術切除視情況與藥物治療之組合。治療可包含投與化學物質(例如放射性同位素、藥物(例如化學治療劑))或遺傳療法(例如抗致癌基因(例如，Rb、DCC、p53等)、顯性負性致癌基因或致癌基因之反義基因)。該等化合物可在其他治療方案之前、與其同時或在其之後投與。舉例而言，可在開始抗細胞增殖療法之前將本發明化合物及/或T細胞活化劑及/或免疫查核點抑制劑投與抗細胞增殖療法(例如，放射療法、化學療法、基因療法、手術切除等)之候選者個體。因此，本發明提供預防性治療方法。 Treatments for use in combination with the compounds of the invention include any antiproliferative, nucleic acid damaging or antineoplastic therapy as disclosed herein or known in the art. For example, anti-cell proliferation or anti-tumor therapy may include radiation therapy or surgical resection optionally in combination with drug therapy. Treatment may involve administration of chemicals (e.g., radioisotopes, drugs (e.g., chemotherapeutics)) or genetic therapy (e.g., anti-oncogenes (e.g., Rb, DCC, p53, etc.), dominant-negative oncogenes, or anti-oncogenes. sense gene). The compounds can be administered before, concurrently with, or after other treatment regimens. For example, a compound of the invention and/or a T cell activator and/or an immune checkpoint inhibitor can be administered to an anti-cell proliferative therapy (e.g., radiation therapy, chemotherapy, gene therapy, surgery) prior to initiating the anti-cell proliferative therapy Candidate individuals for excision, etc.). Accordingly, the present invention provides prophylactic treatment methods.

術語「個體」係指動物，通常係哺乳動物，例如靈長類動物(人類、人猿、長臂猿、黑猩猩、紅毛猩猩、獼猴)、家畜(狗及貓)、農場動物(馬、牛、山羊、綿羊、豬)及實驗動物(小鼠、大鼠、兔、天竺鼠)。個體包括動物疾病模型(例如，具有腫瘤之小鼠)。 The term "individual" refers to an animal, usually a mammal, such as a primate (human, orangutan, gibbon, chimpanzee, orangutan, macaque), livestock (dog and cat), farm animal (horse, cow, goat, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Subjects include animal disease models (eg, mice with tumors).

適於治療之個體包括目前正經歷之彼等或為增殖或分化病症之治療或(例如抗腫瘤療法)之候選者。其他候選者個體包括(例如)具有發生細胞增殖病症之風險之個體。因此，本發明方法可用於治療具有發生細胞增殖病症之風險單尚未展現該病症之明顯症狀之個體。具有風險之個體可經鑑別為具有發生細胞增殖病症之遺傳傾向性或家族史。舉例而言，具有經活化致癌基因或具有腫瘤抑制基因之突變或缺失之個體係候選者個體。因此，具有風險之個體可使用針對遺傳損傷存在之常規遺傳篩選或詢問個體之家族史以確立其具有該病症之風險來鑑別。具有風險之個體之具體實例可為具有家族史或指示癌症傾向性之其他遺傳特徵者，在該遺傳特徵中贅瘤性或抗藥性贅瘤細胞表現CD40。遺傳疾病之具體特定實例係視網膜母細胞瘤，其係由Rb腫瘤抑制基因之缺陷造成的。 Individuals amenable to treatment include those currently undergoing or are candidates for treatment or (eg, antineoplastic therapy) for a proliferative or differentiation disorder. Other candidate individuals include, for example, individuals at risk of developing a cell proliferative disorder. Accordingly, the methods of the invention are useful for treating individuals at risk of developing a cell proliferative disorder who have not yet exhibited overt symptoms of the disorder. Individuals at risk can be identified as having a genetic predisposition or family history of developing a cell proliferative disorder. For example, a phylogenetic candidate individual with an activated oncogene or with a mutation or deletion of a tumor suppressor gene. Thus, at-risk individuals can be identified using conventional genetic screening for the presence of a genetic lesion or by asking the individual's family history to establish their risk for the disorder. A specific example of an individual at risk may be one with a family history or other genetic characteristics indicative of a predisposition to cancer in which neoplastic or drug-resistant neoplastic cells express CD40. A specific example of a genetic disease is retinoblastoma, which is caused by a defect in the Rb tumor suppressor gene.

所投與量通常為「有效量」或「充足量」，其為足以產生期望影響之量。因此，有效量包括以下各項中之一或多者：減少細胞增殖、減少細胞數量、抑制增殖增加、抑制細胞數量增加、增加細胞凋亡，或減少包含增殖細胞之細胞之至少一部分(例如，靶細胞之至少一部分)之存活。因此，例如，倘若期望抑制細胞增殖，則有效量將為可檢測到的減少細胞增殖或增殖細胞數量或增加細胞凋亡或減少細胞存活之量。因此，該量可足以減少靶細胞數量、穩定靶細胞數量或抑制靶細胞數量之增加。舉例而言，倘若病症包含實體腫瘤，則減小腫瘤大小、穩定腫瘤大小或預防腫瘤、腫瘤之至少一部分進一步生長(例如抑制5%至10%之細胞或10%至20%或更多細胞(包含腫瘤團塊)之生長)係令人滿意的臨床終點。倘若病症包含液體腫瘤，則減少腫瘤細胞之數量、穩定腫瘤細胞數量或抑制腫瘤細胞之至少亞群體之腫瘤細胞數量進一步增加(例如抑制5%至10%之細胞或10%至20%或更多之細胞生長)係令人滿意的臨床終點。 The amount administered is generally an "effective amount" or "sufficient amount," which is an amount sufficient to produce the desired effect. Thus, an effective amount includes one or more of: reducing cell proliferation, reducing cell number, inhibiting increased proliferation, inhibiting increased cell number, increasing apoptosis, or reducing at least a portion of cells comprising proliferating cells (e.g., Survival of at least a portion of the target cells). Thus, for example, if inhibition of cell proliferation is desired, an effective amount will be an amount that detectably decreases cell proliferation or the number of proliferating cells or increases apoptosis or decreases cell survival. Accordingly, the amount may be sufficient to reduce the number of target cells, stabilize the number of target cells, or inhibit an increase in the number of target cells. For example, if the disorder comprises a solid tumor, tumor size is reduced, tumor size is stabilized, or tumor, at least a portion of the tumor is prevented from growing further (e.g., inhibiting 5% to 10% of cells, or 10% to 20% or more cells ( Growth comprising tumor mass) is a satisfactory clinical endpoint. If the condition involves a liquid tumor, reducing the number of tumor cells, stabilizing the number of tumor cells, or inhibiting a further increase in the number of tumor cells of at least a subpopulation of tumor cells (eg, inhibiting 5% to 10% of cells or 10% to 20% or more cell growth) is a satisfactory clinical endpoint.

另外，視為有效之量可預防或抑制病況或病症之進展。舉例而言，某些腫瘤在其進展時變得愈來愈具有攻擊性，包括進展至轉移形式。因此，亦視為有效之量可使得減少或預防腫瘤變得愈來愈具有攻擊性或轉移。因此，抑制或預防病症或病況之惡化(亦即穩定病況)係另一令人滿意的臨床終點。 Additionally, an amount considered effective prevents or inhibits the progression of a condition or disorder. For example, certain tumors become increasingly aggressive as they progress, including progression to metastatic forms. Therefore, it is also believed that an effective amount results in the reduction or prevention of tumors becoming more aggressive or metastatic. Therefore, inhibiting or preventing the progression of a disorder or condition (ie, stabilizing the condition) is another desirable clinical endpoint.

檢查含有液體腫瘤之生物試樣(例如，血液或組織試樣)可確立腫瘤細胞團塊或數量是否減少，或是否抑制腫瘤細胞增殖。對於實體腫瘤而言，侵襲及非侵襲成像方法可確定腫瘤大小之減小或腫瘤大小增加之抑制。受體陽性腫瘤之受體計數之降低可用於評價腫瘤細胞增殖之減少或抑制。產激素腫瘤(例如乳癌、睪丸癌或卵巢癌)之激素之量可用於評價腫瘤增殖之減少或抑制。 Examination of a biological sample (eg, a blood or tissue sample) containing liquid tumors can establish whether tumor cell masses or numbers are reduced, or whether tumor cell proliferation is inhibited. For solid tumors, invasive and non-invasive imaging methods can determine a reduction in tumor size or inhibition of an increase in tumor size. A reduction in receptor counts in receptor-positive tumors can be used to assess reduction or inhibition of tumor cell proliferation. Produce Hormones The amount of hormones in tumors such as breast, testicular, or ovarian cancers can be used to assess reduction or inhibition of tumor proliferation.

有效量亦可客觀或主觀地減少或降低與病症或病況相關之症狀之嚴重性或頻次。舉例而言，減少疼痛、噁心或其他不適或增加食欲或主觀幸福感之本發明化合物之量係令人滿意的臨床終點。 An effective amount can also objectively or subjectively reduce or decrease the severity or frequency of symptoms associated with a disorder or condition. For example, the amount of a compound of the invention that reduces pain, nausea or other discomfort or increases appetite or subjective well-being is a desirable clinical endpoint.

有效量亦包括減少用另一方案治療之量(例如，劑量)或頻次，此被認為係令人滿意的臨床終點。舉例而言，利用本發明化合物與T細胞活化劑及/或免疫查核點抑制劑之組合治療之癌症患者可能需要更少的核酸損害治療以便抑制癌症細胞增殖。在此實例中，有效量可包括與在不利用本發明化合物治療下投與之劑量頻率或相比可減少投與個體之核酸損害劑之劑量頻率或量之量。 An effective amount also includes reducing the amount (eg, dose) or frequency of treatment with another regimen, which is considered a satisfactory clinical endpoint. For example, cancer patients treated with combinations of compounds of the invention and T cell activators and/or immune checkpoint inhibitors may require less nucleic acid damaging therapy in order to inhibit cancer cell proliferation. In this example, an effective amount can include an amount that reduces the dosage frequency or amount of a nucleic acid damaging agent administered to an individual as compared to the dosage frequency or amount administered without treatment with a compound of the invention.

可改良個體之病況或治療益處之本發明方法之持續時間可能相對較短，例如改良可持續若干小時、若干天或若干週，或延長更長的時間段，例如數月或數年。有效量無需完全消除病況或病症之任何或所有症狀。因此，當個體之病況在短的或長的時間段內存在主觀或客觀改良(如使用上述準則中之任一者或業內已知適於確定病症或病況之狀態之其他準則所確定)時，可達成有效量之令人滿意的臨床終點。如本文所闡述或業內已知可有效提供一或多個有益效應之量稱作個體之個體病況或「治療益處」之「改良」。 Methods of the invention that improve a subject's condition or benefit from treatment may be of relatively short duration, eg, the improvement may last for hours, days or weeks, or over a longer period of time, eg, months or years. An effective amount need not completely eliminate any or all symptoms of a condition or disorder. Thus, when there is subjective or objective improvement in a subject's condition over a short or long period of time (as determined using any of the above criteria or other criteria known in the art to be suitable for determining the status of a disorder or condition), Satisfactory clinical endpoints can be achieved with effective doses. An amount effective to provide one or more beneficial effects as described herein or known in the art is referred to as an "improvement" of the individual condition or "therapeutic benefit" in the individual.

本發明化合物、T細胞活化劑及免疫查核點抑制劑之有效量可基於動物研究或視情況在人類臨床試驗中來確定。熟習此項技術者將瞭解各種因素均可影響治療具體個體所需要之劑量及時限，包括(例如)個體之整體健康狀況、年齡或性別、病症或病況之嚴重性或狀態、先前治療、對不合意副作用之敏感性、期望之臨床結果及其他病症或病況之存在。該等因素可影響提供足以達成治療益處之量所需要之劑量及時限。劑量方案亦考慮藥物動力學，亦即，醫藥組合物之吸收率、生物利用度、代謝及清除率(例如，參見Egleton(1997)「Bioavailability and transport of peptides and peptide drugs into the brain」Peptides 18：1431-1439；及Langer(1990)Science 249：1527-1533)。另外，劑量或治療方案可針對個體進行特別調整或基於藥物基因體數據進行修改。 Effective amounts of the compounds of the invention, T cell activators, and immune checkpoint inhibitors can be determined based on animal studies or, optionally, in human clinical trials. Those skilled in the art will appreciate that various factors can affect the dosage and duration necessary to treat a particular individual, including, for example, the individual's general health, age or sex, the severity or state of the disorder or condition, previous therapy, whether desirable Sensitivity to side effects, desired clinical outcome, and presence of other disorders or conditions. These factors can affect the dosage and time period necessary to provide an amount sufficient to achieve a therapeutic benefit. Dosage regimens also take into account pharmacokinetics, that is, the rate of absorption, bioavailability, metabolism and clearance of the pharmaceutical composition (see, for example, Egleton (1997) "Bioavailability and transport of peptides and peptide drugs into the brain" Peptides 18: 1431-1439; and Langer (1990) Science 249:1527-1533). In addition, dosage or treatment regimens may be tailored to the individual or modified based on pharmacogenomic data.

因此，化合物、T細胞活化劑及免疫查核點抑制劑可根據達成期望效應之任一方案或途徑，單獨地或作為醫藥組合物以全身方式、以區域方式(例如，針對器官或組織，例如藉由注射至門靜脈中用於治療肝之細胞增殖病症)或以局部方式(例如，直接至腫瘤團塊中)進行投與。化合物及醫藥組合物可作為單一劑量或多個劑量每天(例如，以低劑量)或間歇性地(例如，每隔一天、一週一次等，以較高劑量)進行投與。化合物、T細胞活化劑及免疫查核點抑制劑及醫藥組合物可經由吸入(例如，氣管內)、以經口、靜脈內、動脈內、血管內、鞘內、腹膜內、肌內、皮下、腔內、經皮(例如，局部)、經黏膜(例如，經頰、經膀胱、經陰道、經子宮、經直腸或經鼻)方式藉由多次投與、持續釋放(例如，隨時間逐步灌注)或單次濃注來投與。用於投與藥物之可植入裝置(包括微製作裝置)已為人熟知且亦可用於將本發明化合物遞送至個體。 Thus, the compounds, T cell activators, and immune checkpoint inhibitors may be administered systemically, regionally (e.g., to an organ or tissue, e.g., by Administration is by injection into the portal vein for the treatment of cell proliferative disorders of the liver) or locally (eg, directly into tumor masses). Compounds and pharmaceutical compositions can be administered daily (eg, at low doses) or intermittently (eg, every other day, once a week, etc., at higher doses) as a single dose or in multiple doses. The compounds, T cell activators and immune checkpoint inhibitors, and pharmaceutical compositions can be administered by inhalation (e.g., intratracheal), orally, intravenously, intraarterially, intravascularly, intrathecally, intraperitoneally, intramuscularly, subcutaneously, Intracavitary, transdermal (e.g., topical), transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, or nasal) by multiple administrations, sustained release (e.g., gradually over time perfusion) or as a single bolus. Implantable devices, including microfabricated devices, for administering drugs are well known and can also be used to deliver compounds of the invention to individuals.

以靜脈內方式(IV)投與之化合物、T細胞活化劑及免疫查核點抑制劑在若干小時(通常1小時、3小時或6小時)內可為約0.01mg/hr至約1.0mg/hr，其可在間歇週期下重複一或多個週。特別是在將藥物投與隔離位點且不進入血液流中(例如進入體腔中或進入器官之腔(例如腦脊髓液(CSF)) 中)時，可使用相當高的劑量(例如，高達約10mg/ml)。 Compounds, T cell activators and immune checkpoint inhibitors administered intravenously (IV) may range from about 0.01 mg/hr to about 1.0 mg/hr over several hours (usually 1 hour, 3 hours or 6 hours) , which can be repeated on intermittent cycles for one or more weeks. Especially when the drug is administered to an isolated site and does not enter the bloodstream (eg, into a body cavity or into a lumen of an organ such as cerebrospinal fluid (CSF)) Moderately higher doses (eg, up to about 10 mg/ml) can be used.

因此本發明進一步提供醫藥組合物。該等醫藥組合物可用於在活體內或離體投與個體，及用於例如利用本發明化合物、T細胞活化劑及免疫查核點抑制劑來治療個體。 The present invention therefore further provides pharmaceutical compositions. These pharmaceutical compositions are useful for administering to a subject in vivo or ex vivo, and for treating a subject, for example, with the compounds of the invention, T cell activators and immune checkpoint inhibitors.

如本文所用術語「醫藥上可接受之」及「生理上可接受」包括與醫藥投與相容之溶劑(水性或非水性)、溶液、乳液、分散介質、塗覆劑、等滲劑及吸收促進劑或延遲劑。因此「醫藥組合物」或「醫藥調配物」係指適於在個體中醫藥使用之組合物。醫藥組合物及調配物包括一定量之本發明化合物(例如，有效量之肽或擬肽)、編碼其之核酸、載體或本發明之細胞及醫藥上或生理上可接受之載劑。 As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic agents and absorbents compatible with pharmaceutical administration. accelerator or retarder. A "pharmaceutical composition" or "pharmaceutical formulation" thus refers to a composition suitable for medicinal use in an individual. Pharmaceutical compositions and formulations include an amount of a compound of the invention (eg, an effective amount of a peptide or peptidomimetic), a nucleic acid encoding it, a vector or a cell of the invention, and a pharmaceutically or physiologically acceptable carrier.

醫藥組合物可經調配與具體投與途徑(全身或局部)相容。因此，醫藥組合物包括適於藉由各種途徑投與之載劑、稀釋劑或賦形劑。 Pharmaceutical compositions can be formulated to be compatible with a particular route of administration (systemic or topical). Accordingly, pharmaceutical compositions include carriers, diluents or excipients suitable for administration by various routes.

調配物或腸內(經口)投與可含於錠劑(包衣或未包衣)、膠囊(硬的或軟的)、微球體、乳液、粉末、顆粒、晶體、懸浮液、糖漿或酏劑中。可使用習用非毒性固體載劑來製備固體調配物，該等載劑包括(例如)醫藥級別之甘露醇、乳糖、澱粉、硬脂酸鎂、糖精鈉、滑石粉、纖維素、葡萄糖、蔗糖、碳酸鎂。亦可將補充性活性化合物(例如，防腐劑、抗細菌劑、抗病毒及抗真菌劑)納入調配物中。液體調配物亦可用於腸內投與。載劑可選自各種油，包括石油、動物油、植物油或合成油，例如，花生油、大豆油、礦物油、芝麻油。適宜醫藥賦形劑包括(例如)澱粉、纖維素、滑石粉、葡萄糖、乳糖、蔗糖、明膠、麥芽、水稻、麵粉、白堊、矽膠、硬脂酸鎂、硬脂酸鈉、單硬脂酸甘油酯、氯化鈉、脫脂奶粉、甘油、丙二醇、水、乙醇。 Formulations or enteral (oral) administration may be contained in tablets (coated or uncoated), capsules (hard or soft), microspheres, emulsions, powders, granules, crystals, suspensions, syrups or in elixirs. Solid formulations can be prepared using conventional nontoxic solid carriers including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, magnesium carbonate. Supplementary active compounds such as preservatives, antibacterial, antiviral and antifungal agents can also be incorporated into the formulations. Liquid formulations can also be used for enteral administration. The carrier can be selected from various oils, including petroleum, animal, vegetable or synthetic oils, for example, peanut oil, soybean oil, mineral oil, sesame oil. Suitable pharmaceutical excipients include, for example, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, monostearate Glycerides, Sodium Chloride, Skim Milk Powder, Glycerin, Propylene Glycol, Water, Ethanol.

用於腸內、非經腸或經黏膜遞送之醫藥組合物包括(例如)水、鹽水、磷酸鹽緩衝鹽水、漢克氏溶液(Hank's solution)、林格氏溶液(Ringer's solution)、右旋糖/鹽水及葡萄糖溶液。調配物可含有輔助物質以接近生理條件，例如緩衝劑、張力調節劑、潤濕劑、清潔劑及諸如此類。添加劑亦可包括其他活性成份(例如殺菌劑或穩定劑)。舉例而言，溶液可含有乙酸鈉、乳酸鈉、氯化鈉、氯化鉀、氯化鈣、去水山梨醇單月桂酸酯或三乙醇胺油酸酯。其他非經腸調配物及方法闡述於以下文獻中：Bai(1997)J.Neuroimmunol.80：65-75；Warren(1997)J.Neurol.Sci.152：31-38；及Tonegawa(1997)J.Exp.Med.186：507-515。非經腸製劑可封裝在由玻璃或塑膠製得之安瓿、拋棄式注射器或多劑量小瓶中。 Pharmaceutical compositions for enteral, parenteral or transmucosal delivery include, for example, water, saline, Phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, and glucose solutions. The formulations may contain auxiliary substances to approximate physiological conditions, such as buffers, tonicity adjusting agents, wetting agents, cleansing agents and the like. Additives may also include other active ingredients (such as bactericides or stabilizers). For example, the solution may contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, or triethanolamine oleate. Other parenteral formulations and methods are described in: Bai (1997) J. Neuroimmunol. 80: 65-75; Warren (1997) J. Neurol. Sci. 152: 31-38; and Tonegawa (1997) J. . Exp. Med. 186:507-515. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

用於真皮內或皮下投與之醫藥組合物可包括無菌稀釋劑，例如水、鹽水溶液、不揮發油、聚乙二醇、甘油、丙二醇或其他合成溶劑；抗細菌劑，例如苄醇或對羥苯甲酸甲酯；抗氧化劑，例如抗壞血酸、麩胱甘肽或亞硫酸氫鈉；螯合劑，例如乙二胺四乙酸；緩衝液，例如乙酸鹽、檸檬酸鹽或磷酸鹽；及調節張力之藥劑，例如氯化鈉或右旋糖。 Pharmaceutical compositions for intradermal or subcutaneous administration may include sterile diluents such as water, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or p-hydroxyl; Methyl benzoate; antioxidants, such as ascorbic acid, glutathione, or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and tonicity-adjusting agents , such as sodium chloride or dextrose.

用於注射之醫藥組合物包括水溶液(其中具有水可溶性)或分散液及用於即時製備無菌可注射溶液或分散液之無菌粉末。對於靜脈內投與，適宜載劑包括生理鹽水、抑菌水、克列莫佛EL^TM(Cremophor EL^TM)(BASF,Parsippany,NJ)或磷酸鹽緩衝鹽水(PBS)。載劑可為溶劑或分散介質，其含有例如水、乙醇、多元醇(例如甘油、丙二醇及液體聚乙二醇及諸如此類)及其適宜混合物。可(例如)藉由使用塗層(例如卵磷脂)、在分散液情形下藉由維持所需粒徑，及藉由使用表面活性劑來維持流動性。抗細菌劑及抗真菌劑包括(例如)對羥苯甲酸鹽、氯丁醇、酚、抗壞血酸及硫柳汞。等滲劑(例如，糖、多元醇(例如甘露醇、山梨醇)、氯化鈉)可包括在組合物中。可包裝所得溶液以供按原樣使用，或可將其凍乾，稍後可將凍乾製劑在投與前與無菌溶液合併。 Pharmaceutical compositions for injection include aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline ⁽ PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the desired particle size in the case of dispersions, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents (eg, sugars, polyalcohols (eg, mannitol, sorbitol), sodium chloride) can be included in the composition. The resulting solution can be packaged for use as is, or it can be lyophilized and the lyophilized preparation can later be combined with a sterile solution prior to administration.

醫藥上可接受之載劑可含有穩定、增加或延遲吸收或清除之化合物。該等化合物包括(例如)碳水化合物，例如葡萄糖、蔗糖或聚葡萄糖；低分子量蛋白質；降低肽之清除率或水解之組合物；或賦形劑或其他穩定劑及/或緩衝液。延遲吸收之藥劑包括(例如)單硬脂酸鋁及明膠。亦可使用清潔劑來穩定或增加或降低醫藥組合物之吸收，包括脂質體載劑。為保護免受消化，可將化合物、T細胞活化劑及/或免疫查核點抑制劑與組合物複合以使得其耐受酸性及酶性水解，或可將化合物、T細胞活化劑及/或免疫查核點抑制劑在抗性適當之載劑(例如脂質體)中複合。保護化合物、T細胞活化劑及免疫查核點抑制劑免受消化之方式已為業內習知(例如，參見Fix(1996)Pharm Res.13：1760-1764；Samanen(1996)J.Pharm.Pharmacol.48：119-135；及美國專利5,391,377，其闡述用於經口遞送治療劑之脂質組合物)。 Pharmaceutically acceptable carriers can contain compounds that stabilize, increase or delay absorption or clearance. Such compounds include, for example, carbohydrates, such as glucose, sucrose, or polydextrose; low molecular weight proteins; compositions that reduce clearance or hydrolysis of peptides; or excipients or other stabilizers and/or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize or to increase or decrease the absorption of pharmaceutical compositions, including liposomal vehicles. To protect from digestion, the compound, T cell activator and/or immune checkpoint inhibitor can be complexed with the composition to render it resistant to acidic and enzymatic hydrolysis, or the compound, T cell activator and/or immune checkpoint inhibitor can be complexed with Checkpoint inhibitors are complexed in an appropriate carrier such as liposomes. Means of protecting compounds, T cell activators and immune checkpoint inhibitors from digestion are well known in the art (see, for example, Fix (1996) Pharm Res. 13:1760-1764; Samanen (1996) J. Pharm. Pharmacol. 48:119-135; and US Patent 5,391,377, which describes lipid compositions for oral delivery of therapeutic agents).

對於經黏膜或經皮投與，在調配物中使用適於待滲透屏障之滲透劑。該等滲透劑通常為業內已知，且包括(例如)經黏膜投與、清潔劑、膽汁鹽及梭鏈孢酸衍生物。經黏膜投與可藉助鼻噴霧劑或栓劑進行(例如，參見Sayani(1996)「Systemic delivery of peptides and proteins across absorptive mucosae」Crit.Rev.Ther.Drug Carrier Syst.13：85-184)。為進行經皮投與，可將活性化合物調配成如業內通常已知之軟膏劑、油膏、凝膠或乳霜。亦可使用貼劑達成經皮遞送系統。 For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be by means of nasal sprays or suppositories (see, eg, Sayani (1996) "Systemic delivery of peptides and proteins across absorbent mucosae" Crit. Rev. Ther. Drug Carrier Syst. 13:85-184). For transdermal administration, the active compounds may be formulated into ointments, salves, gels or creams as generally known in the art. Patches can also be used to achieve transdermal delivery systems.

為進行吸入遞送，可將醫藥調配物以氣溶膠或霧劑之形式投與。對於氣溶膠投與，調配物可以微細形式與表面活性劑及推進劑一起供應。在另一實施例中，將調配物遞送至呼吸組織之裝置係調配物在其中蒸發者。業內已知之其他遞送系統包括乾粉氣溶膠、液體遞送系統、吸入器、噴氣式霧化器及推進劑系統(例如，參見Patton(1998)Biotechniques 16：141-143；Dura Pharmaceuticals,San Diego,CA；Aradigm,Hayward,CA；Aerogen,Santa Clara,CA；及Inhale Therapeutic Systems,San Carlos,CA)。 For delivery by inhalation, the pharmaceutical formulations can be administered in the form of an aerosol or mist. For aerosol administration, the formulations can be supplied in micronized form with surfactants and propellants. in another In one embodiment, the device that delivers the formulation to respiratory tissue is one in which the formulation evaporates. Other delivery systems known in the art include dry powder aerosols, liquid delivery systems, inhalers, jet nebulizers, and propellant systems (see, e.g., Patton (1998) Biotechniques 16:141-143; Dura Pharmaceuticals, San Diego, CA; Aradigm, Hayward, CA; Aerogen, Santa Clara, CA; and Inhale Therapeutic Systems, San Carlos, CA).

可使用生物可降解之生物相容聚合物，例如乙烯乙酸乙烯酯、聚酸酐、聚乙醇酸、膠原、聚原酸酯及聚乳酸。製備該等調配物之方法為熟習此項技術者已知。材料亦可在商業上自Alza Corporation and Nova Pharmaceuticals,Inc獲得。亦可使用脂質體懸浮液(包括使用抗體或病毒外殼蛋白靶向細胞或組織之脂質體)作為醫藥上可接受之載劑。該等可根據業內已知之方法來製備，例如，如以下各項中所闡述：美國專利第4,235,871號；第4,501,728號；第4,522,811號；第4,837,028號；第6,110,490號；第6,096,716號；第5,283,185號；第5,279,833號；Akimaru(1995)Cytokines Mol.Ther.1：197-210；Alving(1995)Immunol.Rev.145：5-31；及Szoka(1980)Ann.Rev.Biophys.Bioeng.9：467)。能夠持續遞送小分子(包括肽)之生物可降解微球體或膠囊或其他生物可降解聚合物構形已為業內習知(例如，參見Putney(1998)Nat.Biotechnol.16：153-157)。可將化合物、T細胞活化劑及免疫查核點抑制劑納入膠束內(例如，參見Suntres(1994)J.Pharm.Pharmacol.46：23-28；Woodle(1992)Pharm.Res.9：260-265)。肽可附接至脂質單層或雙層之表面。舉例而言，肽可附接至含有醯肼-PEG-(二硬脂醯基磷脂醯基)乙醇胺之脂質體(例如，參見Zalipsky(1995)Bioconjug.Chem.6：705-708)。或者，可使用脂質膜(例如平面脂質膜)或完整細胞(例如紅血球)之細胞膜中之任一形式。含有脂質體及脂質之調配物可藉由任何方式來遞送，包括(例如)靜脈內、經皮(例如，參見Vutla(1996)J.Pharm.Sci.85：5-8)、經黏膜或經口投與。 Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are known to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to cells or tissues using antibodies or viral coat proteins) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known in the art, for example, as set forth in: US Patent Nos. 4,235,871; 4,501,728; 4,522,811; 4,837,028; 6,110,490; ; No. 5,279,833; Akimaru (1995) Cytokines Mol. Ther. 1: 197-210; Alving (1995) Immunol. Rev. 145: 5-31; and Szoka (1980) Ann. Rev. Biophys. Bioeng. 9: 467 ). Biodegradable microspheres or capsules or other biodegradable polymeric configurations capable of sustained delivery of small molecules, including peptides, are known in the art (eg, see Putney (1998) Nat. Biotechnol. 16:153-157). Compounds, T cell activators, and immune checkpoint inhibitors can be incorporated into micelles (see, for example, Suntres (1994) J. Pharm. Pharmacol. 46:23-28; Woodle (1992) Pharm. Res. 9:260-28. 265). Peptides can be attached to the surface of lipid monolayers or bilayers. For example, peptides can be attached to liposomes containing hydrazine-PEG-(distearoylphosphatiyl)ethanolamine (see, eg, Zalipsky (1995) Bioconjug. Chem. 6:705-708). Alternatively, lipid membranes (e.g. planar lipid membranes) or Any form of the cell membrane of an intact cell such as a red blood cell. Formulations containing liposomes and lipids can be delivered by any means, including, for example, intravenously, transdermally (see, e.g., Vutla (1996) J. Pharm. Sci. 85:5-8), transmucosally or transmucosally. Oral vote.

醫藥上可接受之調配物可納入約1%至99.9%之活性成份(例如，肽或擬肽、T細胞活化劑或免疫查核點抑制劑)。醫藥組合物可藉由習用熟知滅菌技術來滅菌，或可經無菌過濾。 Pharmaceutically acceptable formulations may incorporate from about 1% to 99.9% active ingredient (eg, peptide or peptidomimetic, T cell activator or immune checkpoint inhibitor). Pharmaceutical compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered.

其他醫藥調配物及遞送系統為業內已知且可用於本發明之方法及組合物中(例如，參見Remington’s Pharmaceutical Sciences(1990)第18版，Mack Publishing Co.,Easton,PA；The Merck Index(1996)第12版，Merck Publishing Group,Whitehouse,NJ；Pharmaceutical Principles of Solid Dosage Forms,Technonic Publishing Co.,Inc.,Lancaster,Pa.,(1993)；及Poznansky等人，Drug Delivery Systems,R.L.Juliano編輯，Oxford,N.Y.(1980)，第253頁至第315頁) Other pharmaceutical formulations and delivery systems are known in the art and can be used in the methods and compositions of the invention (see, e.g., Remington's Pharmaceutical Sciences (1990) 18th Edition, Mack Publishing Co., Easton, PA; The Merck Index (1996 ) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993); and Poznansky et al., Drug Delivery Systems, RL Juliano ed., Oxford , NY (1980), pp. 253 to 315)

醫藥調配物可以單位劑量形式調配以便於投與且達成劑量均勻性。如本文所用「單位劑型」係指用於投與欲治療個體之物理上離散之單位劑量；每一單元均含有產生期望效應之預定量之化合物與醫藥載劑或賦形劑之組合。 Pharmaceutical formulations can be formulated in dosage unit form for ease of administration and uniformity of dosage. "Unit dosage form" as used herein refers to physically discrete unit dosages intended to treat the subject; each unit containing a predetermined quantity of the compound to produce the desired effect in combination with a pharmaceutical carrier or excipient.

本文使用以下縮寫： This article uses the following abbreviations:

Cha：環己基-丙胺酸 Cha : cyclohexyl-alanine

Phe-2,3,4,5,6-F：氟化物在苯丙胺酸之苯基殘基上之位置2,3,4,5,6處 Phe-2,3,4,5,6-F : Fluoride at positions 2,3,4,5,6 on the phenyl residue of phenylalanine

F：氟化物 F : Fluoride

Bpa：苯甲醯基-苯丙胺酸 Bpa : benzoyl-phenylalanine

Nal(2)：2-萘基-丙胺醯基 Nal(2) : 2-naphthyl-propanylamino

Ala(3-Bzt)：(3-苯并噻吩基)-丙胺酸 Ala(3-Bzt) : (3-benzothienyl)-alanine

Nal(1)：1-萘基-丙胺醯基 Nal(1) : 1-naphthyl-propanylamino

Dph：二苯基-丙胺酸 Dph : diphenyl-alanine

Ala(tBu)：第三丁基-丙胺醯基 Ala(tBu) : tertiary butyl-acrylamide

Cys(tBu)：第三丁基-半胱胺酸 Cys(tBu) : tertiary butyl-cysteine

Phe-3,4,5-F：氟化物在苯丙胺酸之苯基上之位置3,4,5處 Phe-3,4,5-F : Fluoride at positions 3, 4, and 5 on the phenyl group of phenylalanine

Phe-4CF3：CF3在苯丙胺酸之苯基殘基上之位置4處 Phe-4CF3 : position 4 of CF3 on the phenyl residue of phenylalanine

Phe-3Br,4Cl,5Br：溴化物在苯丙胺酸之苯基上之位置3處，氯化物在位置4處，且溴化物在位置5處 Phe-3Br,4Cl,5Br : bromide at position 3, chloride at position 4, and bromide at position 5 on the phenylalanine

Phe-4Cl：氯化物在苯丙胺酸之苯基上之位置4處 Phe-4Cl : Chloride at position 4 on the phenyl group of phenylalanine

分別P1、P2、P3、P4、P5、P6等，及(P1、P2、P3、P4、P5、P6等)；及P7、P8、P9、P10、P11、P12等，及(P7、P8、P9、P10、P11、P12等)：P1、P2、P3、P4、P5、P6等之鄰接序列；及P7、P8、P9、P10、P11、P12。 P1, P2, P3, P4, P5, P6, etc., and (P1, P2, P3, P4, P5, P6, etc.); and P7, P8, P9, P10, P11, P12, etc., and (P7, P8, P9, P10, P11, P12, etc.) : contiguous sequences of P1, P2, P3, P4, P5, P6, etc.; and P7, P8, P9, P10, P11, P12.

除非另有定義，否則本文所用之所有技術及科學術語皆具有與熟習本發明所屬領域技術者通常所理解之含義相同的含義。儘管在本發明之實踐或測試中可使用類似或等效於本文所述之方法及材料的方法及材料，但本文闡述適宜方法及材料。 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

本文所引用之所有公開案、專利及其他參考文獻之全部內容皆以引用方式併入。倘若出現衝突，則以本說明書(包括定義)為準。 All publications, patents, and other references cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

除非上下文另外明確指出，否則本文所用單數形式「一(a、an)」、「該」及「係(is)」包括複數個指示物。因此，例如，對「化合物」之提及包括複數種化合物，且對「殘基」或「胺基酸」之提及包括提及一或多種殘基及胺基酸。 As used herein, the singular forms "a, an", "the" and "is" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of compounds and reference to "a residue" or "amino acid" includes reference to one or more residues and amino acids.

已闡述眾多本發明實施例。然而，將理解，可在不背離本發明之精神及範圍之情況下作出各種修改。因此，以下實例意欲闡釋但並非限制在申請專利範圍中闡述之本發明之範圍。 A number of embodiments of the invention have been described. However, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of the invention set forth in the claims.

實例example 實例1Example 1

此實例闡述材料及若干方法。此實例亦闡述經分析肽/擬肽之序列。 This example illustrates the materials and several methods. This example also illustrates the sequences of the analyzed peptides/peptidemimetics.

化學品及試劑 博來黴素係購自Wako Pure Chemical Co.(Osaka,Japan)，且將其溶解於蒸餾水中至10mg/ml。碘化丙啶(PI)及阿德力黴素係購自Sigma(St.Louis,MO)。 Chemicals and Reagents Bleomycin was purchased from Wako Pure Chemical Co. (Osaka, Japan), and it was dissolved in distilled water to 10 mg/ml. Propidium iodide (PI) and adriamycin were purchased from Sigma (St. Louis, MO).

細胞培養 在37℃/5% CO₂下在補充有10%胎牛血清(IBL：Immuno-Biological Laboratories,Gunma,Japan)之RPMI 1640(Sigma)中培養人類T細胞白血病源性細胞系Jurkat。在37℃/5% CO₂下在具有10%胎牛血清之DMEM中培養人類胰臟癌源性細胞系MIAPaCa2。 Cell Culture The human T-cell leukemia-derived cell line Jurkat was cultured at 37°C/5% CO ₂ in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum (IBL: Immuno-Biological Laboratories, Gunma, Japan). The human pancreatic cancer-derived cell line MIAPaCa2 was cultured in DMEM with 10% fetal bovine serum at 37°C/5% _CO2 .

細胞週期分析 藉由如Kawabe(1997)Nature 385：454-458所闡述之流式細胞術分析經博來黴素或阿德力黴素處理之細胞之細胞週期狀態。簡言之，在4℃下再懸浮200萬個細胞並在200μl克裡尚氏溶液(Krishan’s solution)(0.1%檸檬酸鈉、50μg/ml PI、20μg/ml RNase A及0.5% NP-40)中培育1hr，並藉由流式細胞術FACScan^TM(Beckton Dickinson,Mountain View,CA)利用程式CELLQuest^TM(Beckton Dickinson)進行分析。 Cell cycle analysis Cell cycle status of bleomycin- or adriamycin-treated cells was analyzed by flow cytometry as described in Kawabe (1997) Nature 385:454-458. Briefly, 2 million cells were resuspended at 4°C and dissolved in 200 μl of Krishan's solution (0.1% sodium citrate, 50 μg/ml PI, 20 μg/ml RNase A, and 0.5% NP-40) Incubate for 1 hr and analyze by flow cytometry FACScan ^TM (Beckton Dickinson, Mountain View, CA) using the program CELLQuest ^TM (Beckton Dickinson).

實例2Example 2

此實例闡述指示各種肽之G2廢除活性及各種序列排列對活性之效應(包括降低序列長度之效應)之數據。 This example sets forth data indicating the G2 abrogating activity of various peptides and the effect of various sequence permutations on activity, including the effect of reducing sequence length.

使用人類白血病源性Jurkat細胞系實施G2查核點廢除之流式細胞術分析。簡言之，利用各種劑量之肽/擬肽及40μg/ml博來黴素將經培養細胞處理24hr。利用碘化丙啶對細胞之DNA進行染色並藉由流式細胞術進行分析。該等結果概述於表2中。 Flow cytometric analysis of G2 checkpoint abolition was performed using the human leukemia-derived Jurkat cell line. Briefly, cultured cells were treated with various doses of peptides/peptidomimetics and 40 μg/ml bleomycin for 24 hr. Cellular DNA was stained with propidium iodide and analyzed by flow cytometry. The results are summarized in Table 2.

在用於對抗博來黴素處理之Jurkat細胞時每一肽/擬肽之劑量反應曲線顯示於圖1、5、6、7、8、11及12中；Y軸指示處理後24hr之G2/M Jurkat細胞%。 Dose-response curves for each peptide/peptidomimetic when used against bleomycin-treated Jurkat cells are shown in Figures 1, 5, 6, 7, 8, 11 and 12; M Jurkat Cell %.

使用經秋水仙鹼(5μg/ml或0.5μg/ml)及各種劑量之肽/擬肽處理24hr之人類T細胞白血病Jurkat細胞系實施化合物對M期查核點廢除之流式細胞術分析(圖12)。對細胞之DNA進行染色並藉由如上文所闡述之流式細胞術進行分析。結果亦概述於表2中。 Flow cytometric analysis of compounds for M-phase checkpoint abolition was performed using human T-cell leukemia Jurkat cell lines treated with colchicine (5 μg/ml or 0.5 μg/ml) and various doses of peptides/peptidomimetics for 24 hrs (Figure 12 ). Cells were stained for DNA and analyzed by flow cytometry as described above. The results are also summarized in Table 2.

當用於對抗秋水仙鹼處理之Jurkat細胞時每一肽/擬肽之劑量反應曲線顯示於圖2及14中；Y軸指示處理後24hr之G2/M Jurkat細胞%。 Dose response curves for each peptide/peptidomimetic when used against colchicine-treated Jurkat cells are shown in Figures 2 and 14; the Y-axis indicates % of G2/M Jurkat cells 24 hr after treatment.

表2. 誘導G2查核點廢除或副作用之化合物之劑量第1欄：(代號) 第2欄：在單獨使用時出現副作用(μM) 第3欄：G2廢除劑量(μM) 第4欄：在與秋水仙鹼一起使用時出現副作用(μM)

Table 2. Doses of Compounds Inducing G2 Checkpoint Abolition or Side Effects Column 1: (Code) Column 2: Adverse Effects When Used Alone (μM) Column 3: G2 Abolition Dose (μM) Column 4: In combination with Side effects when colchicine is used together (μM)

「在單獨使用時出現副作用」指示產生Jurkat細胞週期紊亂之肽/擬肽劑量，亦即，出現顯著量之SubG1細胞(死亡細胞)或每一者之DNA含量變化大於平時之細胞。舉例而言，在FACS分析中G1細胞通常展現尖峰，但在處理後，當經細胞週期受到擾亂時，峰變得更寬且更低，指示不正確細胞週期進展或細胞死亡之開始。「G2廢除劑量」指示在與40μg/ml博來黴素一起處理24小時後產生可檢測到之G2查核點廢除活性之肽/擬肽劑量。「在與秋水仙鹼一起使用時出現副作用」指示在與5μg/ml秋水仙鹼一起處理24小時後產生Jurkat細胞週期紊亂之肽/擬肽劑量。 "Occurrence of side effects when used alone" indicates a dose of the peptide/peptidomimetic that produced a Jurkat cell cycle disturbance, ie, a significant number of SubG1 cells (dead cells) or cells in which the DNA content of each changed more than usual. For example, G1 cells usually exhibit a sharp peak in FACS analysis, but after treatment, when perturbed through the cell cycle, the peak becomes broader and lower, indicating incorrect cell cycle progression or the onset of cell death. "G2 Abolishing Dose" indicates the dose of peptide/peptidomimetic that produces detectable G2 checkpoint abrogating activity after 24 hours of treatment with 40 μg/ml bleomycin. "Adverse effects when used with colchicine" indicates the dose of peptide/peptidomimetic that produced Jurkat cell cycle disturbances after 24 hours of treatment with 5 μg/ml colchicine.

在各種細胞系中研究與順鉑組合時CBP501之G2查核點廢除活性。簡言之，將順鉑(3μg/ml)及CBP501(0.4μM、2μM及10μM)同時添加至細胞培養物中，將該細胞培養物在37℃以及5% CO₂下培育3hr。吸出培養基，添加不含該等化合物之新鮮培養基並將細胞再培育45hr。使用胰蛋白酶-EDTA溶液收穫細胞(包括浮游細胞)，與克裡尚氏溶液一起培育，並如先前所闡述藉由流式細胞術分析DNA含量。該等結果概述於表3中。除HUVEC以外，陰影突顯表示G2群體顯著喪失且subG1群體有所增加之細胞系，指示CBP501可引起G2查核點廢除及對順鉑之敏化。至少高達50μM之CBP501未敏化HUVEC細胞(其為具有正常G1查核點之細胞)之觀察結果指示CBP501對G2查核點係特異的而非非特異的。 The G2 checkpoint abrogating activity of CBP501 in combination with cisplatin was studied in various cell lines. Briefly, cisplatin (3 μg/ml) and CBP501 (0.4 μM, 2 μM and 10 μM) were simultaneously added to cell cultures, which were incubated at 37° C. and 5% CO ₂ for 3 hr. The medium was aspirated, fresh medium without the compounds was added and the cells were incubated for an additional 45 hr. Cells (including planktonic cells) were harvested using trypsin-EDTA solution, incubated with Crischan's solution, and analyzed for DNA content by flow cytometry as previously described. The results are summarized in Table 3. Except for HUVEC, cell lines with significant loss of G2 population and gain of subG1 population are highlighted by shading, indicating that CBP501 can cause G2 checkpoint abolition and sensitization to cisplatin. The observation of CBP501-unsensitized HUVEC cells (which are cells with a normal G1 checkpoint) at least up to 50 μΜ indicates that CBP501 is specific rather than non-specific for the G2 checkpoint.

研究不同劑量之各種化合物對經博來黴素(Bleo)或阿德力黴素(ADR)處理之人類胰臟癌源性細胞系MIAPaCa2之G2查核點廢除活性。簡言之，將細胞與化合物及博來黴素(10μg/ml)或阿德力黴素(1μg/ml)一起培育3小時。更換培養基並再培育21小時。藉由碘化丙啶對所收穫細胞之DNA進行染色並如先前所闡述利用流式細胞術分析。在圖3中，sub-G1細胞群體之%指示為死亡細胞。結果指示CBP501可以劑量依賴性方式使MIAPaCa2細胞對博來黴素及阿德力黴素敏感。 The G2 checkpoint abolition activity of various compounds at different doses on the human pancreatic cancer-derived cell line MIAPaCa2 treated with bleomycin (Bleo) or adriamycin (ADR) was studied. In short, Cells were incubated with compound and either bleomycin (10 μg/ml) or adriamycin (1 μg/ml) for 3 hours. The medium was changed and incubated for an additional 21 hours. DNA from harvested cells was stained by propidium iodide and analyzed by flow cytometry as previously described. In Figure 3, the % of the sub-G1 cell population is indicated as dead cells. The results indicated that CBP501 could sensitize MIAPaCa2 cells to bleomycin and adriamycin in a dose-dependent manner.

圖4A及4C係利用其中一個胺基酸殘基與另一者不同之肽對實施之G2查核點廢除活性之概述。如上文所闡述使用博來黴素處理之Jurkat細胞分析該等肽之G2查核點廢除活性。圖4B係利用其中一個胺基酸殘基與另一者不同之肽對實施之M查核點廢除活性及/或非特異性毒性分析之概述。如上文所闡述使用秋水仙鹼處理之Jurkat細胞分析該等肽之M查核點廢除活性及/或非特異性毒性。 Figures 4A and 4C are summaries of G2 checkpoint abrogation activity performed using peptide pairs in which one amino acid residue differs from the other. The peptides were assayed for G2 checkpoint abrogating activity using bleomycin-treated Jurkat cells as described above. Figure 4B is a summary of M checkpoint abolishing activity and/or non-specific toxicity assays performed using peptide pairs in which one amino acid residue differs from another. The peptides were assayed for M checkpoint abolishing activity and/or non-specific toxicity using colchicine-treated Jurkat cells as described above.

研究不同劑量之各種富精胺酸序列對經博來黴素處理之細胞之G2查核點廢除活性。簡言之，將肽以0.2μg/ml、0.39μg/ml、0.78μg/ml、1.56μg/ml、3.125μg/ml、6.25μg/ml、12.5μg/ml、25μg/ml及50μg/ml添加至具有博來黴素(40μg/ml)之Jurkat細胞培養基中。隨後在24小時後收穫細胞，利用克裡尚氏溶液染色，並如先前所闡述利用流式細胞術分析。在圖5中繪製G2/M細胞%(Y軸)對肽劑量(X軸)之圖形。該等數據指示與具有更少或更大數量殘基之序列相比，「(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：137)」富鹼性殘基序列係最好的序列。 The G2 checkpoint abolishing activity of various doses of various arginine-rich sequences on bleomycin-treated cells was investigated. Briefly, peptides were added at 0.2 μg/ml, 0.39 μg/ml, 0.78 μg/ml, 1.56 μg/ml, 3.125 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml and 50 μg/ml into Jurkat cell culture medium with bleomycin (40 μg/ml). Cells were then harvested 24 hours later, stained with Crischan's solution, and analyzed by flow cytometry as previously described. In Figure 5 the % of G2/M cells (Y-axis) is plotted against peptide dose (X-axis). The data indicate that "(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) is (SEQ ID NO: 137)" The basic residue sequence is the best sequence.

研究不同劑量之不含(D-Bpa)之肽對經博來黴素處理之細胞之G2查核點廢除活性。簡言之，將肽以0.2μg/ml、0.39μg/ml、0.78μg/ml、1.56μg/ml、3.125μg/ml、6.25μg/ml、12.5μg/ml、25μg/ml及50μg/ml添加至具有博來黴素(40μg/ml)之Jurkat細胞培養基中。隨後收穫細胞並如先前所闡述利用流式細胞術分析。在圖6中繪製G2/M細胞%(Y軸)對肽劑量(X軸)之圖形。此結果指示序列(Tyr)(Ser)(Pro)(Trp)(Ser)(Phe-2,3,4,5,6F)(Cha)(SEQ ID NO：138)與序列(Bpa)(Ser)(Trp)(Ser)(Phe-2,3,4,5,6F)(Cha)(SEQ ID NO：139)具有相當的G2查核點廢除活性。 The G2 checkpoint abolishing activity of different doses of peptides without (D-Bpa) on bleomycin-treated cells was studied. Briefly, peptides were added at 0.2 μg/ml, 0.39 μg/ml, 0.78 μg/ml, 1.56 μg/ml, 3.125 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml and 50 μg/ml into Jurkat cell culture medium with bleomycin (40 μg/ml). Cells were subsequently harvested and utilized as previously described Flow cytometry analysis. In Figure 6 the % of G2/M cells (Y-axis) is plotted against peptide dose (X-axis). This result indicates that the sequence (Tyr)(Ser)(Pro)(Trp)(Ser)(Phe-2,3,4,5,6F)(Cha) (SEQ ID NO: 138) is similar to the sequence (Bpa)(Ser) (Trp)(Ser)(Phe-2,3,4,5,6F)(Cha) (SEQ ID NO: 139) has comparable G2 checkpoint abolition activity.

研究不同劑量之富精胺酸及富離胺酸序列對經博來黴素處理之細胞之G2查核點廢除活性。簡言之，將肽以指示劑量(X軸)添加至具有博來黴素(40μg/ml)之Jurkat細胞培養基中。隨後收穫細胞並如先前所闡述利用流式細胞術進行分析。在圖7中繪製G2/M細胞%(Y軸)對肽劑量之圖形。結果指示Arg序列似乎提供較富鹼性胺基酸序列之Lys序列更好之活性且Gln並非序列之功能所必需的。 To study the G2 checkpoint abolition activity of different doses of arginine-rich and lysine-rich sequences on cells treated with bleomycin. Briefly, peptides were added to Jurkat cell culture medium with bleomycin (40 μg/ml) at the indicated doses (X-axis). Cells were then harvested and analyzed by flow cytometry as previously described. In Figure 7 the % of G2/M cells (Y-axis) is plotted against peptide dose. The results indicated that the Arg sequence seemed to provide better activity than the Lys sequence of the basic amino acid rich sequence and that Gln was not necessary for the function of the sequence.

研究富精胺酸區之位置有所變化之序列之G2查核點廢除活性。簡言之，將肽以所指示劑量(X軸)添加至具有博來黴素(40μg/ml)之Jurkat細胞培養基中並保持24小時。隨後收穫細胞並如先前所闡述利用流式細胞術進行分析。在圖8中繪製G2/M細胞%(Y軸)對肽劑量之圖形。 The G2 checkpoint abrogating activity of sequences with changes in the position of the arginine-rich region was investigated. Briefly, peptides were added to Jurkat cell culture medium with bleomycin (40 μg/ml) at the indicated doses (X-axis) for 24 hours. Cells were then harvested and analyzed by flow cytometry as previously described. In Figure 8 the % of G2/M cells (Y-axis) is plotted against peptide dose.

數據指示肽之G2廢除活性不因改變富精胺酸區之位置而顯著改變。另外，CBP501可溶於水，而CBP511不可溶於水。此差異對具體藥物遞送系統可能有利，乃因一些系統優選水不溶性化合物。 The data indicate that the G2 abrogating activity of the peptide was not significantly altered by changing the position of the arginine-rich region. In addition, CBP501 is soluble in water while CBP511 is insoluble in water. This difference may be advantageous for particular drug delivery systems, as some systems prefer water-insoluble compounds.

圖9圖解說明利用其中僅一個胺基酸殘基在各對之間有所不同之各種肽對實施之分析之概述。使用如所闡述之博來黴素處理之Jurkat細胞分析該等肽之G2查核點廢除活性。 Figure 9 illustrates a summary of analyzes performed with various pairs of peptides in which only one amino acid residue differs between pairs. The peptides were assayed for G2 checkpoint abrogating activity using bleomycin-treated Jurkat cells as described.

每一胺基酸之大小、電荷及疏水性決定序列如何有效地適合於靶分子。肽或擬肽之側鏈可自由移動，因此即使具有一或兩個不利側鏈，肽或擬肽亦可適合靶分子之袋或槽。該概述指示每一側鏈均具有較佳大小，其表明每一側鏈之靶蛋白質之結合區(袋或槽)之大小。舉例而言，具有環結構(例如苯、吲哚及環己烷)之側鏈決定G2廢除或M廢除及/或非特異性毒性之強度；參見圖9及圖4，其中大於5員之環結構影響G2廢除活性(在P1及P2處具有中等大小可增加G2廢除活性，而過大之結構(P1、P5及P6)增加M廢除及/或非特異性毒性)。 The size, charge and hydrophobicity of each amino acid determine how efficiently the sequence fits on the target molecule. The side chains of a peptide or peptidomimetic are free to move, so even with one or two unfavorable side chains, a peptide or peptidomimetic can fit into a pocket or groove of a target molecule. This overview indicates that each side chain has an optimal size, its The size of the binding region (pocket or groove) of the target protein is indicated for each side chain. For example, side chains with ring structures such as benzene, indole, and cyclohexane determine the strength of G2 abolition or M abolition and/or non-specific toxicity; see Figure 9 and Figure 4, where rings with more than 5 members Structure influences G2 abrogation activity (intermediate size at P1 and P2 increases G2 abrogation activity, while oversized structures (P1, P5 and P6) increase M abolition and/or non-specific toxicity).

沒有環結構之側鏈似乎沒有作用。因此，為獲得較佳活性，P1、P2、P4及P6處之環結構之大小應適當，且期望在P3及P5處沒有環結構或小於6員之環結構。P1、P2及P6之適當環來自1員至6員環，兩個環藉助融合而具有5個或6個成員。對於P4，大小適當之環係兩個環之融合，該兩個環中之每一者均為5員或6員的。因此，對於P1，Cha或Nal(2)似乎最適合；對於P2，Phe-2,3,4,5,6F、Phe-3,4,5F或Phe-CF3似乎最好。該等側鏈之大小指示在此區發生相互作用之靶分子中存在兩個袋或單一較大袋。對於P3及P5，諸如Ser或Pro等小側鏈係可接受的且諸如Arg等較大側鏈亦可接受，指示在靶分子之此區中不存在袋，因此側鏈可能僅與靶標相對。然而，環結構可能使得肽或擬肽能夠與另一分子(即，不同於靶分子)發生相互作用，此又可增加副作用。對於P6，Bpa或Ser-Tyr似乎好於單獨Tyr或較小側鏈，指示較深槽水平地位於靶標中。基於P4之殘基之大小，在靶標中亦可存在用於P4之淺且較寬之袋。 Side chains without ring structures appear to have no effect. Therefore, in order to obtain better activity, the size of the ring structures at P1, P2, P4 and P6 should be appropriate, and it is desirable that there is no ring structure or a ring structure with less than 6 members at P3 and P5. Appropriate loops for P1, P2 and P6 are from 1 to 6 membered loops, with both loops having 5 or 6 members by fusion. For P4, the appropriate size loop is the fusion of two loops, each of which is 5- or 6-membered. Therefore, for P1, Cha or Nal(2) seemed to be the most suitable; for P2, Phe-2,3,4,5,6F, Phe-3,4,5F or Phe-CF3 seemed to be the best. The size of these side chains indicates the presence of two pockets or a single larger pocket in the target molecule interacting in this region. For P3 and P5, small side chains such as Ser or Pro are acceptable and larger side chains such as Arg are also acceptable, indicating that there is no pocket in this region of the target molecule, so the side chain may only be opposite the target. However, the loop structure may enable the peptide or peptidomimetic to interact with another molecule (ie, different from the target molecule), which in turn can increase side effects. For P6, Bpa or Ser-Tyr appeared to be better than Tyr alone or the smaller side chain, indicating that the deeper groove is located horizontally in the target. Based on the size of the residues of P4, there may also be a shallow and wider pocket for P4 in the target.

如所闡述使用Jurkat及博來黴素分析以下肽。肽之序列為如下：CBP501，(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO：80)；CBP700，(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：96)；CBP701，(d-Arg)(d-Arg) (d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：97)；CBP702，(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：98)；及CBP703，(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO：99)。結果指示CBP700、701、702、703儘管短於其他例示之肽，但其保留與具有顯著G2查核點廢除活性之其他肽相當之G2查核點廢除活性(圖11)。 The following peptides were analyzed using Jurkat and bleomycin as described. The sequence of the peptide is as follows: CBP501, (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 80); CBP700, (d-Arg)(d-Arg) (d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 96); CBP701, (d-Arg)(d-Arg) (d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO: 97); CBP702, (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d- Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO:98); and CBP703, (d-Arg)(d-Arg)(d-Arg)(d-Bpa) (d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 99). The results indicated that CBP700, 701 , 702, 703, although shorter than the other exemplified peptides, retained comparable G2 checkpoint abrogating activity to other peptides with significant G2 checkpoint abrogating activity (Figure 11).

在由CBP501引起之G2查核點廢除活性與非特異性毒性(M查核點廢除)之間實施比較。簡言之，對於G2查核點廢除活性及非特異性毒性分別利用40μg/ml博來黴素或0.5μg/ml秋水仙鹼處理Jurkat細胞。如先前所闡述藉由流式細胞術分析每一經處理細胞中之DNA量。數據指示G2查核點係以CBP501劑量依賴性方式廢除，同時在高達50μM之肽下不存在非特異性毒性，如藉由未變化之M期停滯之細胞之百分比所確定(圖12)。 A comparison was performed between G2 checkpoint abrogation activity and non-specific toxicity (M checkpoint abolition) induced by CBP501. Briefly, Jurkat cells were treated with 40 μg/ml bleomycin or 0.5 μg/ml colchicine for G2 checkpoint abrogating activity and non-specific toxicity, respectively. The amount of DNA in each treated cell was analyzed by flow cytometry as previously described. The data indicated that the G2 checkpoint was abolished in a CBP501 dose-dependent manner with the absence of non-specific toxicity up to 50 μΜ of the peptide, as determined by the unchanged percentage of cells arrested in M phase (Figure 12).

實例3Example 3

此實例闡述各種肽之肽/擬肽激酶抑制活性及血清穩定性分析。 This example illustrates the peptide/peptidomimetic kinase inhibitory activity and serum stability assays of various peptides.

由於兩種激酶Chk1及Chk2對於G2查核點機制至關重要，故實施兩種酶之激酶抑制分析。使用「PepTag^(R)Non-Radioactive Protein Kinase Assays」,Promega，根據公司之方案實施活體外激酶抑制分析，只是使用經純化CHK2激酶替代PKC。經純化PKC係購自Upstate Biotechnology,Inc。該等結果顯示於表4A中。 Since two kinases, Chk1 and Chk2, are critical to the G2 checkpoint mechanism, a kinase inhibition assay for both enzymes was performed. Using "PepTag ^(R) Non-Radioactive Protein Kinase Assays", Promega, carried out in vitro kinase inhibition assays according to the company's protocol, except that purified CHK2 kinase was used instead of PKC. Purified PKC was purchased from Upstate Biotechnology, Inc. The results are shown in Table 4A.

活體外激酶抑制分析係由CycLex,Co.Ltd.,Nagano,Japan實施。簡言之，使用具有組胺酸標籤之桿狀病毒源性重組體人類全長Chk1或融合有GST之大腸桿菌(E.Coli)源性重組體人類全長Chk2作為激酶。使用大腸桿菌源性重組體GST-Cdc25C(胺基酸167-267)作為受質。反應條件為20mM Hepes-KOH(pH7.5)、1mM DTT、80μg/ml BSA、10mM MgCl₂及50mM ATP，在30℃下，保持60min。利用酶聯免疫分析藉由抗Cdc25C-磷酸化S216抗體檢測GST-Cdc25C上絲胺酸216之磷酸化。該等結果顯示於表4B中。 In vitro kinase inhibition assays were performed by CycLex, Co. Ltd., Nagano, Japan. Briefly, baculovirus-derived recombinant human full-length Chk1 with a histidine tag or E. coli-derived recombinant human full-length Chk2 fused with GST were used as kinases. Escherichia coli-derived recombinant GST-Cdc25C (amino acids 167-267) was used as a substrate. The reaction conditions were 20 mM Hepes-KOH (pH 7.5), 1 mM DTT, 80 μg/ml BSA, 10 mM MgCl ₂ and 50 mM ATP, at 30° C. for 60 min. Phosphorylation of Serine 216 on GST-Cdc25C was detected by ELISA with an anti-Cdc25C-phosphorylated S216 antibody. The results are shown in Table 4B.

數據指示在高於G2廢除劑量之劑量下發生Chk1及Chk2激酶抑制(由CBP500、501、505、506、603引起之G2廢除之IC50皆小於1μM)。該等結果表明該等肽除抑制Chk1/2分子以外亦具有作用機制。或者，肽可能累積在細胞內使得其在細胞內之濃度大於在周圍培養基中之濃度。 The data indicated that inhibition of Chk1 and Chk2 kinases occurred at doses higher than the G2 abolishing dose (IC50 for G2 abolition by CBP500, 501, 505, 506, 603 were all less than 1 μΜ). These results suggest that these peptides also have a mechanism of action other than inhibition of Chk1/2 molecules. Alternatively, the peptide may accumulate intracellularly such that its concentration within the cell is greater than that in the surrounding medium.

在小鼠及人類血清中實施血清分析來確定肽之穩定性。簡言之，在37℃下將肽(10mM或2.5mM)與剛剛製備之人類血清一起培育1hr。在37℃下將CBP501(10mM)與剛剛製備之小鼠血清一起培育1hr。用有或沒有肽及博來黴素(40μg/ml)之血清處理Jurkat細胞並培育24hr。如先前所闡述藉由流式細胞術測定G2期細胞之群體。_>藉由比較經處理血清之G2細胞%及利用培養基處理之肽、博來黴素及Jurkat細胞產生之標準曲線(表5A)測定血清處理之肽之殘餘G2查核點廢除活性。在利用乙醇處理去保護之後利用HPLC測定殘餘CBP501量(表5B)。數據指示，具有d型胺基酸之肽(例如CBP501及CBP603)在血清中較具有l型胺基酸之肽(例如CBP413)更穩定。 Serum analysis was performed in mouse and human sera to determine peptide stability. Briefly, peptides (10 mM or 2.5 mM) were incubated with freshly prepared human serum for 1 hr at 37°C. CBP501 (10 mM) was incubated with freshly prepared mouse serum for 1 hr at 37°C. Jurkat cells were treated with serum with or without peptide and bleomycin (40 μg/ml) and incubated for 24 hr. The population of cells in G2 phase was determined by flow cytometry as previously described. _> Residual G2 checkpoint abrogating activity of serum-treated peptides was determined by comparing the % of G2 cells of treated serum to a standard curve generated with medium-treated peptides, bleomycin and Jurkat cells (Table 5A). The amount of residual CBP501 was determined by HPLC after deprotection by treatment with ethanol (Table 5B). The data indicate that peptides with d-type amino acids such as CBP501 and CBP603 are more stable in serum than peptides with l-type amino acids such as CBP413.

實例4Example 4

此實例闡述CBP501對經培養細胞之抗細胞增殖活性。此實例亦闡述證實肽/擬肽之活體內活性之數據。 This example illustrates the anti-cell proliferative activity of CBP501 on cultured cells. This example also sets forth data demonstrating the in vivo activity of the peptides/peptidomimetics.

為證實化合物之抗細胞增殖活性，單獨及組合利用CBP501(10μmM)、順鉑(1μg/ml、3μg/ml或9μg/ml)及奧沙利鉑(1μg/ml、3μg/ml或9μg/ml)處理經培養MIAPaCa2人類胰臟癌細胞。簡言之，將細胞以300個細胞/孔平鋪於6孔板中，培育過夜，並利用化合物處理3小時。更換培養基並再培養10天。隨後利用70%甲醇固定細胞，利用0.1%結晶紫染色並進行可視化。群落形成分析結果指示CBP501可使順鉑及奧沙利鉑對抗MIAPaCa2細胞之細胞毒性活性增強。 To confirm the anti-proliferative activity of the compound, CBP501 (10 μmM), cisplatin (1 μg/ml, 3 μg/ml or 9 μg/ml) and oxaliplatin (1 μg/ml, 3 μg/ml or 9 μg/ml) were used alone or in combination. ) treated cultured MIAPaCa2 human pancreatic cancer cells. Briefly, cells were plated in 6-well plates at 300 cells/well, incubated overnight, and treated with compounds for 3 hours. The medium was changed and cultured for another 10 days. Cells were then fixed with 70% methanol, stained with 0.1% crystal violet, and visualized. The results of the colony formation assay indicated that CBP501 could enhance the cytotoxic activity of cisplatin and oxaliplatin against MIAPaCa2 cells.

使用正常人類臍內皮細胞(HUVEC)實施類似研究。由於正常細胞不形成結腸，故將其以3000個細胞/孔而非300個細胞/孔進行平鋪。結果指示肽本身不擾亂正常細胞之生長，且肽亦不加強順鉑及奧沙利鉑對細胞之細胞毒性活性。因此，與對核酸損害治療敏感之高增殖細胞(例如癌細胞)相反，肽似乎不展現對抗經受核酸損害處理之正常細胞之顯著G2廢除活性。結果指示肽在敏化增殖細胞而非抵抗核酸損害治療之正常細胞中具有特異性。 Similar studies were performed using normal human umbilical endothelial cells (HUVEC). Since normal cells do not form colons, they were plated at 3000 cells/well instead of 300 cells/well. The results indicated that the peptide itself did not disturb the growth of normal cells, nor did the peptide potentiate the cytotoxic activity of cisplatin and oxaliplatin on the cells. Thus, in contrast to hyperproliferative cells (such as cancer cells) that are sensitive to nucleic acid damaging therapy, The peptides did not appear to exhibit significant G2 abrogating activity against normal cells subjected to nucleic acid damaging treatment. The results indicate that the peptide is specific in sensitizing proliferating cells but not normal cells resistant to nucleic acid damaging treatment.

實施阿爾瑪藍分析以分析與及不與順鉑一起之CBP501之生長抑制活性。簡言之，以一式兩份方式使MIAPaCa2細胞在96孔板中以2500個細胞/孔暴露於1μM、3μM、10μM、30μM、100μM順鉑或0.22μM、0.67μM、2μM、6μM及18μM有或沒有10μM順鉑之CBP501達3小時。更換培養基並再培育24小時、48小時或72小時。在培育後，將20μl 90%阿爾瑪藍試劑添加至每一孔中並再保持6小時用於藉由螢光強度檢測細胞存活率。使用具有激發530nm及發射590nm之Spectrafluor Plus板讀取器量測螢光強度。計算IC50(表6)。 Alamar blue assay was performed to analyze the growth inhibitory activity of CBP501 with and without cisplatin. Briefly, MIAPaCa2 cells were exposed in duplicate to 1 μM, 3 μM, 10 μM, 30 μM, 100 μM cisplatin or 0.22 μM, 0.67 μM, 2 μM, 6 μM and 18 μM with or CBP501 without 10 μM cisplatin for 3 hours. The medium was changed and incubated for an additional 24 hours, 48 hours or 72 hours. After incubation, 20 μl of 90% alamar blue reagent was added to each well and kept for another 6 hours for detection of cell viability by fluorescence intensity. Fluorescent intensity was measured using a Spectrafluor Plus plate reader with excitation 530 nm and emission 590 nm. IC50s were calculated (Table 6).

此研究指示以莫耳劑量計單獨CBP501對細胞生長之抑制好於順鉑。CBP501在與接近癌症治療所用順鉑之劑量之10μM順鉑組合時可以遠遠更低之劑量抑制細胞生長。此外，CBP501之生長抑制活性長於順鉑；在使用CBP501時72小時時之IC50遠遠好於順鉑。 This study indicates that CBP501 alone inhibits cell growth better than cisplatin on a molar dose basis. CBP501 inhibited cell growth at much lower doses when combined with 10 μM cisplatin at doses close to those used in cancer therapy. In addition, the growth inhibitory activity of CBP501 is longer than that of cisplatin; IC50 at 72 hours when using CBP501 is much better than that of cisplatin.

藉由在腹膜內注射CBP501(40mg/kg)之後的1hr、3hr及6hr對小鼠血清中CBP501進行定量來測定CBP501之活體內半衰期。在利用乙醇處理對自經注射小鼠吸出之小鼠血清進行去保護之後，利用HPLC測定殘餘完整CBP501量(表7)。 The in vivo half-life of CBP501 was determined by quantifying CBP501 in mouse serum 1 hr, 3 hr and 6 hr after intraperitoneal injection of CBP501 (40 mg/kg). After deprotecting mouse sera aspirated from injected mice by ethanol treatment, the amount of residual intact CBP501 was determined by HPLC (Table 7).

為測定對肽之耐受性，利用CBP501(5mg/kg、8mg/kg或10mg/kg)將十隻小鼠之群組靜脈內注射一次或利用CBP501(50mg/kg、80mg/kg或100mg/kg)將其腹膜內注射一次。觀察經注射小鼠一週之存活(表8)。 To determine tolerance to the peptide, groups of ten mice were injected intravenously once with CBP501 (5 mg/kg, 8 mg/kg or 10 mg/kg) or with CBP501 (50 mg/kg, 80 mg/kg or 100 mg/kg/ kg) were injected intraperitoneally once. Survival of the injected mice was observed for one week (Table 8).

為研究化合物之活體內效能，在scid小鼠中皮下植入MIAPaCa2人類胰臟癌細胞。當原發性腫瘤之大小變成0.1cm³(第0天)或更大(例如直徑為7mm或8mm)時開始治療。以單獨或組合方式腹膜內投與CDDP(3mg/kg)及CBP 501(10mg/kg或40mg/kg)。一週三次使用測徑器量測腫瘤大小，且使用以下公式計算體積：重量(mg)=[寬度(mm)2×長度(mm)]/2。繪製每一治療組之平均腫瘤大小對開始治療後的天數的圖形(n=4)(圖10)。 To study the in vivo efficacy of compounds, MIAPaCa2 human pancreatic cancer cells were subcutaneously implanted in scid mice. Treatment begins when the size of the primary tumor becomes 0.1 ^cm3 (day 0) or larger (eg, 7 mm or 8 mm in diameter). CDDP (3 mg/kg) and CBP 501 (10 mg/kg or 40 mg/kg) were intraperitoneally administered alone or in combination. Tumor size was measured using calipers three times a week, and the volume was calculated using the following formula: weight (mg) = [width (mm)2 x length (mm)]/2. The mean tumor size for each treatment group was plotted against the number of days after initiation of treatment (n=4) (Figure 10).

結果指示單獨CBP501治療可抑制活體內人類胰臟癌細胞之生長。結果進一步指示，CBP501可增加順鉑之抗腫瘤活性。 The results indicate that CBP501 treatment alone can inhibit the growth of human pancreatic cancer cells in vivo. The results further indicate that CBP501 can increase the antitumor activity of cisplatin.

實例5Example 5

此實例包括肺癌及使用CBP501之研究之闡述。 This example includes a description of lung cancer and studies using CBP501.

肺癌係西方國家成人癌症死亡之主要原因。在USA中，2009年診斷出219,440例新病例且159,390例死亡係由此疾病造成，佔所有癌症死亡之約29%(例如，參見American cancer society,Cancer Facts & Figures 2009)。所有新肺癌病例之百分之八十七(87%)皆係非小細胞肺癌(NSCLC)組織學，其中存在三種主要類型：腺癌、鱗狀細胞(表皮樣)癌及大細胞癌(American cancer society,Cancer Facts & Figures 2009)。儘管手術技術及組合療法有所改良，但診斷患有NSCLC之患者之預後仍然較差。對於在早期在疾病仍係局部的時檢測出來之病例，五年存活率為47%，但大多數NSCLC患者(68%)(例如，參見AJCC Cancer Staging Manual.Fleming ID編輯，Philadelphia：Lippincott-Raven；2002)經診斷患有需要化學療法之晚期疾病(III期)或轉移疾病(IV期)。患有III期疾病之彼等患者之5年存活率為8.4%且患有IV期者為1.6%，其中大多數患者患有晚期NSCLC，在2年內死於疾病(例如，參見American cancer society,Cancer Facts & Figures2009；AJCC Cancer Staging Manual.,Fleming ID編輯，Philadelphia：Lippincott-Raven；2002)。亟需引入可使患者存活及生活品質顯著改良之新療法。 Lung cancer is the leading cause of cancer death among adults in Western countries. In the USA, 219,440 new cases were diagnosed and 159,390 deaths were attributable to this disease in 2009, accounting for approximately 29% of all cancer deaths (see, eg, American cancer society, Cancer Facts & Figures 2009). Eighty-seven percent (87%) of all new lung cancer cases are from non-small cell lung cancer (NSCLC) tissue cancer, of which there are three main types: adenocarcinoma, squamous cell (epidermoid) carcinoma, and large cell carcinoma (American cancer society, Cancer Facts & Figures 2009). Despite improvements in surgical techniques and combination therapies, the prognosis of patients diagnosed with NSCLC remains poor. For cases detected early, when the disease is still localized, the five-year survival rate is 47%, but the majority of NSCLC patients (68%) (see, eg, AJCC Cancer Staging Manual. Fleming ID ed., Philadelphia: Lippincott-Raven ; 2002) diagnosed with advanced disease (Stage III) or metastatic disease (Stage IV) requiring chemotherapy. The 5-year survival rate of those patients with stage III disease was 8.4% and those with stage IV was 1.6%, with the majority of patients with advanced NSCLC dying of the disease within 2 years (see, for example, American cancer society , Cancer Facts & Figures 2009; AJCC Cancer Staging Manual., edited by Fleming ID, Philadelphia: Lippincott-Raven; 2002). There is an urgent need to introduce new therapies that can lead to significant improvements in patient survival and quality of life.

具有良好體能狀態之患有晚期(IIIb或IV)NSCLC之患者可獲益於化學療法(例如，參見Souquet,PJ.等人，Lancet 342：19-21,1993；Marino,P.等人，Chest 106：861-865,1994；Marino,P.等人，Cancer 76：593-601,1995；Helsing,M.等人，Eur J Cancer 34：1036-1044,1998；Cullen,MH.等人，J Clin Oncol 17：3188-3194,1999；Pfister,DG.等人，J Clin Oncol 22：330-353,2004)。在與單一藥劑或不存在化學療法比較時，化學療法雙聯體已顯示可改良存活(例如，參見Bunn,PA.等人，J Clin Oncol 20：23S-33S,2002)。在晚期NSCLC中目前推薦之一線化學療法方案包括鉑化合物(順鉑[CDDP]或卡鉑)與吉西他濱、長春瑞濱或紫杉烷(太平洋紫杉醇或多西他賽)、伊立替康、依託泊苷、長春鹼及/或培美曲塞之組合作為參考方案(Pfister,DG.等人，J Clin Oncol 22：330-353,2004)。 Patients with advanced (IIIb or IV) NSCLC with good performance status can benefit from chemotherapy (see, for example, Souquet, PJ. et al., Lancet 342:19-21, 1993; Marino, P. et al., Chest 106: 861-865, 1994; Marino, P. et al., Cancer 76: 593-601, 1995; Helsing, M. et al., Eur J Cancer 34: 1036-1044, 1998; Cullen, MH. et al., J Clin Oncol 17:3188-3194, 1999; Pfister, DG. et al., J Clin Oncol 22:330-353, 2004). Chemotherapy doublets have been shown to improve survival when compared to single agents or the absence of chemotherapy (eg, see Bunn, PA. et al., J Clin Oncol 20:23S-33S, 2002). Currently recommended first-line chemotherapy regimens in advanced NSCLC include platinum compounds (cisplatin [CDDP] or carboplatin) with gemcitabine, vinorelbine, or taxanes (paclitaxel or docetaxel), irinotecan, etopol A combination of glycosides, vinblastine and/or pemetrexed was used as a reference protocol (Pfister, DG. et al., J Clin Oncol 22:330-353, 2004).

隨機化試驗已顯示儘管方案在毒性、便利性及成本方面略微不同，但各種鉑雙聯體組合皆具有類似效能。結果發現總體反應率(ORR)在17%與32%之間，中值存活時間為7個月至10個月，且1年存活率為30%至45%(例如，參見Scagliotti,G.等人，Semin Oncol 32：S5-S8,2005；Schiller,JH.等人，N Engl J Med 346：92-98,2002；Scagliotti,G.等人，J Clin Oncol 20：4285-4291,2002；Kelly,K.等人，J Clin Oncol 19：3210-3218,2001；Fossella,F.等人，J Clin Oncol 21：3016-3024,2003)。 Randomized trials have shown that various platinum doublet combinations have similar efficacy, although the regimens differ slightly in toxicity, convenience, and cost. The overall response rate (ORR) was found to be between 17% and 32%, the median survival time was 7 months to 10 months, and the 1-year survival rate was 30% to 45% (for example, see Scagliotti, G. et al. People, Semin Oncol 32:S5-S8, 2005; Schiller, JH. et al., N Engl J Med 346:92-98, 2002; Scagliotti, G. et al., J Clin Oncol 20:4285-4291, 2002; Kelly , K. et al., J Clin Oncol 19:3210-3218, 2001; Fossella, F. et al., J Clin Oncol 21:3016-3024, 2003).

三聯體化學療法之大多數情況迄今尚未進一步延長存活，而是增加了毒性。然而，卡鉑+太平洋紫杉醇+貝伐珠單抗(bevacizumab)之最近研究顯示一些存活益處(例如，參見Sandler,A.等人，N Engl J Med 355：2542-2550,2006)，表明增添具有非重疊毒性之靶向劑可改良雙聯體化學療法。正藉助使用預測抗腫瘤活性之分子標記物積極嘗試最佳化化學療法之益處。預測NSCLC中化學治療劑效能之基因正開始出現(例如，參見Bepler,G.等人，ASCO Educational Book：350-352,2008；Sommers,K.等人，Proc Am Soc Clin Oncol 26 2008)。其中尤其值得注意的是諸如ERCC1、BRCA1/2、RRM1及TS等標記物(表9)。 Most cases of triplet chemotherapy to date have not further prolonged survival but increased toxicity. However, a recent study of carboplatin+paclitaxel+bevacizumab showed some survival benefit (eg, see Sandler, A. et al., N Engl J Med 355:2542-2550, 2006), suggesting that addition has Targeting agents with non-overlapping toxicity may improve doublet chemotherapy. Active attempts are being made to optimize the benefit of chemotherapy through the use of molecular markers that predict antitumor activity. Genes that predict the efficacy of chemotherapeutic agents in NSCLC are beginning to emerge (see, eg, Bepler, G. et al., ASCO Educational Book: 350-352, 2008; Sommers, K. et al., Proc Am Soc Clin Oncol 26 2008). Of particular note are markers such as ERCC1, BRCA1/2, RRM1 and TS (Table 9).

實例6Example 6

此實例包括指示某些人類患者亞群體有利地響應肽及化學治療(核酸損害)劑之組合之數據之闡述。意外地，該等數據顯示針對在利用CBP501治療前每立方毫米血液具有小於10,000個白血球(WBC)計數之非鱗狀非小細胞肺癌(NSCLC)之臨床研究之患者群體之亞組可受益於CBP501之投與。 This example includes a presentation of data indicating that certain subpopulations of human patients respond favorably to combinations of peptides and chemotherapeutic (nucleic acid damaging) agents. Unexpectedly, these data show that a subgroup of the patient population of the clinical study for non-squamous non-small cell lung cancer (NSCLC) with a white blood cell (WBC) count of less than 10,000 per cubic millimeter of blood prior to treatment with CBP501 can benefit from CBP501 of investment.

CBP501係完全由D-胺基酸組成之合成型十二肽(圖13)。其係TAT-S216A之演進型式，在基於DNA含量流式細胞術之分析中，該TAT-S216A針對其因應DNA損害劑之治療而減少G2(4N)細胞之累積之活性經最佳化。 CBP501 is a synthetic dodecapeptide composed entirely of D-amino acids (Figure 13). It is an evolved version of TAT-S216A optimized for its activity in reducing accumulation of G2(4N) cells in response to treatment with DNA damaging agents in DNA content flow cytometry based assays.

已在總共78名患者中執行兩個I期劑量範圍及藥物動力學研究來研究CBP501：CBP501之單一療法研究，其係在第1天、第8天及第15天作為60min的i.v.輸注投與，每4週重複一次；及每3週投與一次之與順鉑組合之療法研究(例如，參見Shapiro,GI.等人，ClinCancer Res.5月15日；17(10)：3431-42,2011)。 Two Phase I dose-ranging and pharmacokinetic studies have been performed to investigate CBP501 in a total of 78 patients: A monotherapy study of CBP501 administered as a 60-min i.v. infusion on Days 1, 8 and 15 , repeated every 4 weeks; and a study of therapy in combination with cisplatin administered every 3 weeks (eg, see Shapiro, GI. et al., Clin Cancer Res. May 15; 17(10):3431-42, 2011).

I期單一藥劑研究(CBP04-01)：此係首次用於人體之單一藥劑I期劑量遞增試驗，其探索在患有晚期實體腫瘤之患者群體中每28天進行三次注射(第1天至第8天至第15天)之方案。總共投與68個週期，每名患者之中值週期數均為2(範圍1至8)。兩名患者達成7個週期之穩定疾病治療，一名診斷為患有胰臟癌且另一名診斷為患有卵巢癌。大多數患者(87%)由於疾病進展中斷了研究。沒有患者由於毒性而中斷(例如，參見Shapiro,GI.等人，ClinCancer Res.5月15日；17(10)：3431-42,2011)。 Phase I single-agent study (CBP04-01): This is the first-in-human phase I dose-escalation study of a single agent, which explores three injections every 28 days (Day 1 to Day 1) in a patient population with advanced solid tumors. 8 days to 15 days). A total of 68 cycles were administered, with a median number of cycles per patient of 2 (range 1 to 8). Two patients achieved 7 cycles of stable disease treatment, one diagnosed with pancreatic cancer and the other diagnosed with ovarian cancer. Most patients (87%) discontinued the study due to disease progression. No patients discontinued due to toxicity (eg, see Shapiro, GI. et al., Clin Cancer Res. May 15;17(10):3431-42, 2011).

CBP501與順鉑(CBP06-01)之組合之I期研究：此I期研究之主要目標係確定CBP501及順鉑在每21天一次組合投與時之MTD及RD。首先以1小時之輸注投與CBP501，隨後在開始治療後2小時投與順鉑。亦根據針對I期單一藥劑研究研發之同一方案給予患者針對過敏反應之預防性治療(氯雷他定(loratadine)、地塞米松(dexamethasone)、雷尼替丁(ranitidine)及苯海拉明(diphenhydramine))。 Phase I Study of Combination of CBP501 and Cisplatin (CBP06-01): The primary objective of this Phase I study was to determine the MTD and RD of CBP501 and Cisplatin when administered in combination every 21 days. CBP501 was administered first as a 1 hour infusion, followed by cisplatin 2 hours after initiation of treatment. Patients were also given prophylactic treatment for anaphylaxis (loratadine, dexamethasone, ranitidine, and diphenhydramine ( diphenhydramine)).

在三個US中心中總共治療48名患者且總共投與182個週期，每名患者之中值週期數均為4(範圍1至13)。在3.6mg/m²至36.4mg/m²之劑量範圍內探索CBP501。所研究之最高劑量量為CBP501 36.4mg/m²及順鉑75mg/m²。在此劑量量下，2/6的患者經歷過敏反應，此係由研究者來判斷，且此可作為劑量限制(3級)。MTD視為稍低於該劑量限制之劑量量，其為CBP501 24.3mg/m²及順鉑75mg/m²。在若干患者中均記錄到活性之提示(例如，參見Shapiro,GI.等人，ClinCancer Res.5月15日；17(10)：3431-42,2011)。 A total of 48 patients were treated in three US centers and a total of 182 cycles were administered, with a median number of cycles per patient of 4 (range 1 to 13). CBP501 was explored in a dose range of 3.6 mg/m ² to 36.4 mg/m ² . The highest doses studied were CBP501 36.4 mg/m ² and cisplatin 75 mg/m ² . At this dose, 2/6 patients experienced anaphylaxis, which was at the discretion of the investigator and this could serve as a dose limiter (Grade 3). The MTDs are considered to be slightly below the dose limiting doses of 24.3 mg/ ^m2 for CBP501 and 75 mg/ ^m2 for cisplatin. Suggestions of activity were noted in several patients (eg, see Shapiro, GI. et al., Clin Cancer Res. May 15;17(10):3431-42, 2011).

順鉑(順式-二氨絡二氯鉑)(無機鉑配位錯合物)優先在DNA之鳥嘌呤及腺嘌呤殘基之N7位置處反應以形成各種單功能及雙功能加合物。該等加合物因阻止需要使兩個DNA鏈分離之各種細胞過程(例如複製及轉錄)而促成藥物之細胞毒性。 Cisplatin (cis-diamminedichloroplatinum), an inorganic platinum coordination complex, reacts preferentially at the N7 position of guanine and adenine residues of DNA to form various monofunctional and bifunctional adducts. These adducts contribute to the cytotoxicity of the drug by preventing various cellular processes that require the separation of the two DNA strands, such as replication and transcription.

順鉑由於其充分抵抗睪丸癌及卵巢癌之抗贅瘤活性而已在臨床上針對各種腫瘤得到評價。由於順鉑之批准，其一直以來為關鍵化學治療劑且已單獨或與其他抗贅瘤劑組合廣泛使用。亦已知順鉑可在呈現其他腫瘤類型(例如肺癌、膀胱癌及頭頸癌)之患者中賦予實質姑息性效應，且其包括在該等疾病所使用之大多數化學療法方案中。 Cisplatin has been evaluated clinically against various tumors due to its antineoplastic activity well against testicular and ovarian cancers. Since its approval, cisplatin has been a key chemotherapeutic agent and has been widely used alone or in combination with other antineoplastic agents. Cisplatin is also known to confer substantial palliative effects in patients presenting with other tumor types such as lung, bladder and head and neck cancers, and is included in most chemotherapy regimens used for these diseases.

培美曲塞二鈉係具有獨特的6-5經稠合吡咯并[2,3-d]嘧啶核之結構上新穎之抗葉酸劑，且其抑制參與合成細胞生長及分裂所必需受質(例如胸腺嘧啶核苷酸合酶、二氫葉酸還原酶及甘胺醯胺核糖核苷酸甲醯基轉移酶)之葉酸依賴性酶之功能(例如，參見Taylor,EC.等人，J Med Chem 35：4450-4454,1992；Schultz,RM.等人，Anticancer Res 19：437-443,1999)。 Pemetrexed disodium has a unique 6-5 fused pyrrolo[2,3-d]pyrimidine core structure Novel antifolate agent, and it inhibits the synthesis of essential substances for cell growth and division (such as thymidine synthase, dihydrofolate reductase and glycylamide ribonucleotide formyltransferase) Function of folate-dependent enzymes (see, eg, Taylor, EC. et al., J Med Chem 35:4450-4454, 1992; Schultz, RM. et al., Anticancer Res 19:437-443, 1999).

已在臨床試驗中在眾多種腫瘤類型(包括肺、乳房、結腸、胸膜、胰臟、胃、膀胱、頭頸及子宮頸)中證實培美曲塞具有活性。培美曲塞與順鉑之組合係在2004年2月4日由FDA批准用於治療患有MPM之患者，該患者之疾病不可切除或換言之其不為治癒性手術之候選者。 Pemetrexed has demonstrated activity in clinical trials in a wide variety of tumor types, including lung, breast, colon, pleura, pancreas, stomach, bladder, head and neck, and cervix. The combination of pemetrexed and cisplatin was approved by the FDA on February 4, 2004 for the treatment of patients with MPM whose disease is not resectable or in other words not candidates for curative surgery.

在患有NSCLC之化學療法初治患者之II期研究中，培美曲塞與順鉑或卡鉑之組合所產生效能結果與其他鉑雙聯體相當(例如，參見Scagliotti,G.等人，Clincancer Res 11：690-696,2005；Zinner R.等人，Cancer 104：2449-2456,2005；Manegold,C.等人，Ann Oncol 11：435-440,2000；Shepherd,FA.等人，Cancer 92：595-600,2001)。另外，培美曲塞具有優良安全性概況及便利投與時間表。 In a phase II study of chemotherapy-naïve patients with NSCLC, the combination of pemetrexed and cisplatin or carboplatin produced comparable efficacy results to other platinum doublets (see, for example, Scagliotti, G. et al., Clincancer Res 11: 690-696, 2005; Zinner R. et al., Cancer 104: 2449-2456, 2005; Manegold, C. et al., Ann Oncol 11: 435-440, 2000; Shepherd, FA. et al., Cancer 92: 595-600, 2001). In addition, pemetrexed has an excellent safety profile and a convenient dosing schedule.

最近隨機化之III期研究在非劣性設計試驗中比較在每3週經順鉑加吉西他濱或順鉑加培美曲塞治療長達六個週期之1725名患有III期或IV期NSCLC之化學療法初治患者之間的整體存活(OS)(例如，參見Scagliotti,G.等人，J Clin Oncol 26：3543-3551,2008；Pimentel,F.等人，Proc Am Soc Clin Oncol 26(第I部分/共II部分)：448s,2008,(增刊15S)(摘要)#448s)。順鉑加培美曲塞之OS並不次於順鉑加吉西他濱(兩種治療之中值存活均為10.3個月)。在患有腺癌(n=847；分別12.6個月及10.9個月)及大細胞癌組織學(n=153；分別10.4個月及6.7個月)之患者中，順鉑加培美曲塞之OS在統計學上優於順鉑/吉西他濱。對於順鉑加培美曲塞，3級或4級嗜中性球減少症、貧血及血小板減少症、發熱性嗜中性球減少症及脫髪之比率顯著低於順鉑/吉西他濱治療組，但3級或4級噁心更為普遍。 A recent randomized phase III study compared chemotherapy in 1725 patients with stage III or IV NSCLC treated with cisplatin plus gemcitabine or cisplatin plus pemetrexed every 3 weeks in a non-inferiority design trial for up to six cycles. Overall survival (OS) among therapy-naïve patients (see, for example, Scagliotti, G. et al., J Clin Oncol 26:3543-3551, 2008; Pimentel, F. et al., Proc Am Soc Clin Oncol 26 (Part I Part/Part II): 448s, 2008, (Supplement 15S) (Abstract) #448s). The OS of cisplatin plus pemetrexed was not inferior to that of cisplatin plus gemcitabine (median survival of both treatments was 10.3 months). Cisplatin plus pemetrexed OS in Statistically better than cisplatin/gemcitabine. For cisplatin plus pemetrexed, the rates of grade 3 or 4 neutropenia, anemia and thrombocytopenia, febrile neutropenia, and hair loss were significantly lower than those for cisplatin/gemcitabine, But grade 3 or 4 nausea was more common.

患者及方法：Patients and Methods:

臨床研究設計：開放標籤、多中心、II期隨機化、兩組的比較研究。該方案評估有或沒有CBP501之全劑量順鉑及培美曲塞。將患者以1：1比率隨機化至培美曲塞、順鉑及CBP501(A組)或培美曲塞及順鉑(B組)。根據疾病之基線狀態(IIIb對IV)、腦轉移之存在及患者對於貝伐珠單抗療法是否合格對隨機化進行分層。 Clinical study design: open-label, multicenter, phase II randomized, comparative study between two groups. This regimen evaluates full-dose cisplatin and pemetrexed with or without CBP501. Patients were randomized in a 1:1 ratio to pemetrexed, cisplatin and CBP501 (arm A) or pemetrexed and cisplatin (arm B). Randomization was stratified according to baseline disease status (IIIb vs. IV), presence of brain metastases, and patient eligibility for bevacizumab therapy.

研究者/試驗位置：在USA、俄羅斯(Russia)、加拿大(Canada)、巴西(Brazil)、阿根廷(Argentina)及秘魯(Peru)中接近40個中心。 Investigator/Trial Location: Nearly 40 centers in USA, Russia, Canada, Brazil, Argentina and Peru.

研究目標：Research objectives:

主要：為在患有局部晚期(具有惡性胸膜滲出液或心包滲出液之IIIB期)或轉移(IV期)非鱗狀NSCLC之患者中比較有或沒有CBP501之順鉑及培美曲塞之效能、無進展存活。 Primary: To compare the efficacy of cisplatin and pemetrexed with or without CBP501 in patients with locally advanced (stage IIIB with malignant pleural or pericardial effusion) or metastatic (stage IV) non-squamous NSCLC , Progression-free survival.

次要：為描述研究方案之安全性概況及除無進展存活以外之效能參數(例如整體存活)。 Secondary: To describe the safety profile of the study protocol and performance parameters other than progression-free survival (eg overall survival).

研究群體：Research groups: 納入凖則：Inclusion rules:

1.在開始任何研究特異性程序之前獲得簽名知情同意書。 1. Obtain signed informed consent prior to commencing any study-specific procedures.

2.在組織學上或細胞學上證實非鱗狀非小細胞肺癌(NSCLC)之診斷，不適於根治性切除術，具有胸膜或心包滲出液之IIIB期或IV期，該研究群體未接受先前化學療法或其他全身性治療。 2. Histologically or cytologically confirmed diagnosis of non-squamous non-small cell lung cancer (NSCLC), not suitable for radical resection, stage IIIB or stage IV with pleural or pericardial effusion, this study group has not accepted previous Chemotherapy or other systemic treatments.

3.根據實體腫瘤中之反應評估準則(RECIST)，存在至少一個一維可量測之病灶。 3. According to the Response Evaluation Criteria in Solid Tumors (RECIST), there is at least one one-dimensional measurable lesion.

4.男性或女性患者為至少18歲。 4. Male or female patients are at least 18 years old.

5. ECOG體能狀態(PS)：0至1。 5. ECOG Performance Status (PS): 0 to 1.

6.預期壽命>3個月。 6. Life expectancy > 3 months.

7.若在研究藥劑之第一劑量之前完成

3週，則容許先前局部放射療法。 7. If completed before the first dose of study drug

3 weeks prior local radiation therapy was allowed.

8.對於用於疼痛控制之現有骨病灶，容許伴隨的姑息性放射療法。 8. Concomitant palliative radiation therapy is permitted for existing bone lesions for pain management.

9.若在研究藥劑之第一劑量之前至少4週實施先前手術，則先前手術係容許的，且患者應完全恢復。 9. Prior surgery is permitted if performed at least 4 weeks prior to the first dose of study drug, and the patient should recover fully.

10.器官功能充足，包括以下： 10. Adequate organ function, including the following:

骨髓：白血球(WBC)計數

4×10⁹/L，絕對嗜中性球計數(ANC)

1.5×10⁹/L，血小板計數

100×10⁹/L，血紅素

9g/dL肝：膽紅素

1.5×正常值上限(ULN)，天冬胺酸胺基轉移酶(AST/SGOT)及丙胺酸胺基轉移酶(ALT/SGPT)

2.5×ULN(或若存在肝轉移，則

5×ULN)，INR

1.5×ULN，白蛋白

3.0g/dL。 Bone Marrow: White Blood Cell (WBC) Count

4×10 ⁹ /L, absolute neutrophil count (ANC)

1.5×10 ⁹ /L, platelet count

100×10 ⁹ /L, hemoglobin

9g/dL liver: bilirubin

1.5 x upper limit of normal (ULN), aspartate aminotransferase (AST/SGOT) and alanine aminotransferase (ALT/SGPT)

2.5×ULN (or if liver metastases are present, then

5×ULN), INR

1.5 x ULN, albumin

3.0g/dL.

腎：血清肌酸酐

1.5mg/dL或肌酸酐清除率

45mL/min(根據Cockroft-Gault公式計算)。 Kidney: Serum creatinine

1.5 mg/dL or creatinine clearance

45mL/min (calculated according to the Cockroft-Gault formula).

11.具有生育潛能之女性患者必須具有陰性懷孕測試，且在研究之前的4週及研究藥物最後劑量後的4個月使用如研究者批准之至少一種形式之避孕。出於此研究之目的，生育潛能定義為：「除處於絕經後至少一年或以手術方式不育者以外之所有女性患者」。 11. Female patients of reproductive potential must have a negative pregnancy test and use at least one form of contraception as approved by the investigator for 4 weeks prior to the study and 4 months after the last dose of study drug. For the purposes of this study, reproductive potential was defined as "all female patients except those who were at least one year postmenopausal or who were surgically infertile."

12.在研究期間及研究藥物最後劑量後的4個月，男性患者必須使用研究者批准之一種形式之障壁避孕。 12. During the study period and 4 months after the last dose of the study drug, male patients must use the study drug A form of barrier contraception approved by researchers.

13.能夠與治療及隨訪合作。 13. Able to cooperate with treatment and follow-up.

排除準則：Exclusion criteria:

1.在進入研究之前實施放射療法之骨髓超過30%。 1. More than 30% of the bone marrow underwent radiotherapy before entering the study.

2.腫瘤試樣中存在神經內分泌特徵。 2. Presence of neuroendocrine features in tumor samples.

3.先前治療利用化學療法、新穎生物療法(小分子、抗體)、免疫療法。 3. Prior treatment utilized chemotherapy, novel biological therapies (small molecules, antibodies), immunotherapy.

4.不存在可量測病灶。 4. There are no measurable lesions.

5.在研究者看來，存在持續或活動性感染、症狀性鬱血性心臟衰竭、不穩定性心絞痛、症狀性或控制不佳之心律不整、不受控之血栓或出血性病症或任何其他嚴重的不受控醫學病症。 5. In the investigator's opinion, there is persistent or active infection, symptomatic congestive heart failure, unstable angina, symptomatic or poorly controlled arrhythmia, uncontrolled thrombosis or bleeding disorder, or any other serious Uncontrolled medical condition.

6.在進入研究5年內存在另一惡性病之任一先前歷史(除治癒之皮膚基底細胞癌或治癒之子宮頸原位癌以外)。 6. Any prior history of another malignancy within 5 years of study entry (except cured basal cell carcinoma of the skin or cured carcinoma in situ of the cervix).

7.存在可妨礙患者順從性之任一顯著中樞神經系統(CNS)或精神異常。 7. Presence of any significant central nervous system (CNS) or psychiatric abnormalities that could impede patient compliance.

8.存在外周神經病變>1級(根據NCI-CTCAE版本3)之證據。 8. Evidence of peripheral neuropathy > grade 1 (according to NCI-CTCAE version 3).

9.在進入研究前的28天內利用任一其他研究藥劑治療，或參與另一臨床試驗。 9. Use any other research drug treatment within 28 days before entering the study, or participate in another clinical trial.

10.懷孕或哺乳患者或具有分娩潛能之任一患者未使用充足避孕。 10. Pregnant or breastfeeding patients or any patient with childbirth potential who have not used adequate contraception.

11.存在已知之HIV、HBV、HCV感染。 11. Known HIV, HBV, HCV infection.

12.存在症狀性腦轉移。具有腦轉移之患者必須：在完成確定性療法後在局部療法(手術或輻射)後至少2週內具有穩定神經狀態，且在進入研究前已中斷皮質類固醇之使用達1週。 12. There are symptomatic brain metastases. Patients with brain metastases must: have a stable neurological status for at least 2 weeks after local therapy (surgery or radiation) after completion of definitive therapy and have been off corticosteroids for 1 week prior to study entry.

不存在可能混淆神經及其他AE之評估之神經功能障礙。 There were no neurological deficits that could confound the assessment of neurological and other AEs.

13.無法或不願意服用葉酸、維生素B12或皮質類固醇。 13. Unable or unwilling to take folic acid, vitamin B12, or corticosteroids.

14.阿斯匹林(aspirin)或除劑量

1.3克/天之阿斯匹林外之其他非類固醇消炎劑不能中斷達5天時段(對於諸如吡羅昔康(piroxicam)之長效藥劑為8天時段)。 14. Aspirin or detoxification

NSAIDs other than aspirin at 1.3 g/day should not be interrupted for a period of 5 days (or a period of 8 days for long-acting agents such as piroxicam).

15.存在顯著重量損失(在前6週期間重量損失

10%體重)。 15. Presence of significant weight loss (weight loss during the previous 6 weeks

10% body weight).

16.存在臨床上顯著的(藉由體檢)第三空間體液聚集物，例如腹水或胸膜滲出液，其在進入研究前無法藉由引流或其他程序得到控制。 16. Presence of clinically significant (by physical examination) third space fluid accumulations, such as ascites or pleural effusion, which could not be controlled by drainage or other procedures prior to study entry.

患者之數量：Number of patients:

總共治療195名患者，其中利用CBP501、順鉑及培美曲塞(A組)治療97名患者且利用培美曲塞及順鉑(B組)治療98名患者。 A total of 195 patients were treated, of which 97 patients were treated with CBP501, cisplatin and pemetrexed (arm A) and 98 patients were treated with pemetrexed and cisplatin (arm B).

研究藥物：Study drug:

調配物：用於注射之CBP501係含有包含CBP501肽乙酸鹽(肽基單元)之無菌凍乾粉之單一劑量小瓶(20mg)中提供。為進行投與，在5%右旋糖注射液(USP)中重構小瓶內容物，並將其添加至具有5%右旋糖注射液(USP)之100mL i.v.袋中。 Formulation: CBP501 for injection is supplied in single-dose vials (20 mg) containing sterile lyophilized powder comprising CBP501 peptide acetate (peptidyl unit). For administration, reconstitute the contents of the vial in 5% Dextrose Injection (USP) and add it to a 100 mL iv bag with 5% Dextrose Injection (USP).

培美曲塞：使用培美曲塞之市售調配物，其中在20mL注射用0.9%氯化鈉溶液中進行重構，然後將其稀釋至100mL。 Pemetrexed: A commercial formulation of pemetrexed was used, reconstituted in 20 mL of 0.9% sodium chloride solution for injection, which was then diluted to 100 mL.

順鉑：使用順鉑之市售調配物且在250mL用於投與之生理鹽水中稀釋。 Cisplatin: A commercial formulation of cisplatin was used and diluted in 250 mL of normal saline for administration.

劑量方案及投與途徑：Dosage regimen and route of administration:

每3週在同一天(第1天)投與CBP501、培美曲塞及順鉑，並保持最多六個週期。將週期視為3週(21天)。 Administer CBP501, pemetrexed, and cisplatin on the same day (Day 1) every 3 weeks for up to six cycles. Think of the cycle as 3 weeks (21 days).

A組Group A

1.以1小時之i.v.輸注投與CBP501 25mg/m²。 1. Administer CBP501 25 mg/m ² by 1 hour iv infusion.

2.在CBP501輸注之後立即以10分鐘之i.v.輸注投與培美曲塞500mg/m²。 2. Administer pemetrexed 500 mg/ ^m2 as a 10-minute iv infusion immediately after CBP501 infusion.

3.在培美曲塞輸注之後立即以1小時之i.v.輸注投與順鉑75mg/m²。 3. Administer cisplatin 75 mg/ ^m2 as a 1 hour iv infusion immediately after the pemetrexed infusion.

B組Group B

1.以10分鐘之i.v.輸注投與培美曲塞500mg/m²。 1. Administer pemetrexed 500 mg/m ² as an iv infusion over 10 minutes.

2.在培美曲塞輸注之後立即以1小時之i.v.輸注投與順鉑75mg/m²。 2. Administer cisplatin 75 mg/ ^m2 as a 1 hour iv infusion immediately after the pemetrexed infusion.

每一組合均經由中樞或外周靜脈通路來投與。 Each combination is administered via central or peripheral venous access.

預防性治療：Preventive treatment: 所有經招募患者皆接受：All recruited patients receive:

1.維生素補充物：每天指導所有患者服用低劑量口服葉酸製劑或具有葉酸之多種維生素劑。在培美曲塞之第一劑量之前的7天時段期間必須已服用至少5個葉酸日劑量，且在整個療程期間及培美曲塞之最後劑量之後的21天應繼續投藥。所建議之葉酸劑量在350μg至1000μg之範圍內。患者亦必須在培美曲塞第一劑量之前的一週期間接受一次(1)維生素B12之肌內注射且此後每3個週期接受一次。後續的維生素B12注射可在培美曲塞之同一天給予。維生素B12之劑量為1000μg。 1. Vitamin supplements: Instruct all patients to take low-dose oral folic acid preparations or multivitamins containing folic acid every day. At least 5 daily doses of folic acid must have been taken during the 7-day period prior to the first dose of pemetrexed and should continue throughout the course of treatment and for 21 days after the last dose of pemetrexed. Suggested folic acid dosages range from 350 μg to 1000 μg. Patients must also receive one (1) intramuscular injection of vitamin B12 during the week prior to the first dose of pemetrexed and every 3 cycles thereafter. Subsequent vitamin B12 injections can be given on the same day as pemetrexed. The dosage of vitamin B12 is 1000μg.

2.在治療投與日之前及之後，每天兩次經口投與4mg地塞米松。 2. Before and after the treatment administration day, 4 mg of dexamethasone was orally administered twice a day.

3.預防性止吐治療：根據標準治療中心方案由5HT3拮抗劑+類固醇組成。視需要給予患者其他口服止吐劑。 3. Prophylactic antiemetic therapy: according to the standard treatment center protocol consisting of 5HT3 antagonists + steroids. Administer additional oral antiemetics to the patient as needed.

˙在沒有心血管損害之患者中建議以下水份(hydration)方案。在研究者中心常規投與之類似方案可能已實施： ˙The following hydration regimen is recommended in patients without cardiovascular compromise. A similar protocol to that routinely administered at the Investigator Center may have been implemented:

1.患者接受總共1.5公升至2.0公升具有20mEq KCl/公升及1g MgSO₄/公升之水份(5%右旋糖或½生理鹽水)，此係以500mL/小時運行。 1. The patient receives a total of 1.5 liters to 2.0 liters of water (5% dextrose or ½ normal saline) with 20 mEq KCl/liter and 1 g MgSO ₄ /liter, which is run at 500 mL/hour.

2.在患者接受1小時之水合物輸注之後，藉由IV推注投與12.5g甘露醇。 2. Administer 12.5 g of mannitol by IV bolus after the patient receives a 1 hour hydrate infusion.

3.然後經1小時輸注順鉑輸注(以1mg/mL在生理鹽水中混合)，同時繼續水份輸注。 3. Then infuse cisplatin infusion (mixed in saline at 1 mg/mL) over 1 hour while continuing the water infusion.

4.若有必要維持尿排出量在水份之持續時間內為250mL/小時，則投與額外甘露醇(12.5-50.0g，藉由IV推注)。 4. Administer additional mannitol (12.5-50.0 g by IV bolus) if necessary to maintain urine output at 250 mL/hour for a sustained period of water.

˙對於經CBP501(A組)治療之患者，推薦其接受以下預防性方案以降低由組織胺釋放引起之症狀之發病率及嚴重性： ˙For patients treated with CBP501 (Group A), it is recommended to accept the following preventive regimens to reduce the incidence and severity of symptoms caused by histamine release:

1.在每一CBP501輸注前，投與苯海拉明(DPH)50mg IV及雷尼替丁50mg IV(或另一組織胺H2拮抗劑)。 1. Administer diphenhydramine (DPH) 50 mg IV and ranitidine 50 mg IV (or another histamine H2 antagonist) prior to each CBP501 infusion.

2.在投與CBP501之前(第-1天)、在投與CBP501當天(第0天)及在投與CBP501之後(第1天)PO投與氯雷他定(10mg)。 2. PO administration of loratadine (10 mg) before CBP501 administration (Day -1), on the day of CBP501 administration (Day 0) and after CBP501 administration (Day 1).

每名患者研究時段之持續時間：Duration of study session per patient:

除非早期觀察到以下各項中之任一者，否則患者將接受最多留個週期之研究治療： Patients will receive a maximum of six cycles of study treatment unless any of the following are observed early:

˙疾病進展 Disease progression

˙不可接受之毒性 ˙Unacceptable toxicity

˙撤銷同意 ˙Revocation of consent

˙嚴重違反方案 ˙Serious violation of the plan

˙治療延遲>2週(在潛在或感知到的患者益處之情形下除外) ˙Treatment delayed >2 weeks (except in cases of potential or perceived patient benefit)

在治療中斷後，患者每8週進行隨訪直至疾病進展或開始其他全身性抗癌療法為止，且然後每6個月進行隨訪直至死亡為止。 Following treatment interruption, patients were followed every 8 weeks until disease progression or initiation of other systemic anticancer therapy, and then every 6 months until death.

知情同意書informed consent

在執行任何研究特異性篩選程序之前，研究者向患者充分解釋研究之目的及方法以及任何預期效應及不利反應。為患者提供資訊表且給予其充足時間及機會來諮詢試驗之詳情並決定是否參與。患者及其論述知情同意書之人簽署同意書並註明其日期。 Before performing any study-specific screening procedures, the investigators fully explained the purpose and methods of the study and any expected effects and adverse reactions to the patients. Provide patients with an information sheet and give them sufficient time and opportunity to consult the details of the trial and decide whether to participate. The consent form was signed and dated by the patient and the person discussing the informed consent form.

研究者解釋患者可完全自由的拒絕進入研究或在任一時間及出於任一原因退出該研究。類似地，研究者及/或贊助商可出於安全性或投與原因而在任一時間自由領回患者。根據目前CFR(21，部分312D、50及56)及ICH(ICH E6 1997)GCP指南及Declaration of Helsinki,1964(如2004年在東京(Tokyo)所闡明)解釋保護患者之人權所必需之任何其他要求。 The investigator explains that patients are completely free to refuse to enter the study or to withdraw from the study at any time and for any reason. Similarly, investigators and/or sponsors are free to bring back patients at any time for safety or administration reasons. Any other interpretation necessary to protect the human rights of patients in accordance with current CFR (21, Parts 312D, 50 and 56) and ICH (ICH E6 1997) GCP guidelines and Declaration of Helsinki, 1964 (as stated in Tokyo (Tokyo) 2004) Require.

患者數量之分配Allocation of the number of patients

集中管控至治療組之患者隨機化及分配。 Randomization and assignment of patients to treatment groups was centrally managed.

統計學分析：Statistical analysis:

使用Cox比例危險模型來估計2個治療組之間PFS之危險比(HR)。該模型包括治療組因素以及隨機化分層因素。探索以下共變量：年齡、性別、種族(高加索人(Caucasian)/非高加索人)、先前手術/程序(是/否)、先前放射療法(是/否)、x射線詮釋(正常/異常)、ECG詮釋(正常/異常)、骨掃描(正常/異常)及診斷時間。若可產生更好模型擬合，則可藉由指定2類或若干類值將任何連續變量(例如年齡及診斷至研究治療之時間)轉化為類別變量。使用逐步回歸演算法使用以下準則輸入探索性變量：變量必須在0.25位準下顯著以輸入模型中且在0.15位準下顯著以保持於模型中。對於最終模型，連同95% CI一起提供危險比之點估計值。 Cox proportional hazards models were used to estimate the hazard ratio (HR) for PFS between the 2 treatment groups. The model included treatment group factors as well as randomization stratification factors. The following covariates were explored: age, sex, race (Caucasian/non-Caucasian), previous surgery/procedure (yes/no), previous radiation therapy (yes/no), x-ray interpretation (normal/abnormal), ECG interpretation (normal/abnormal), bone scan (normal/abnormal) and time of diagnosis. Any continuous variable (such as age and time from diagnosis to study treatment) can be converted to a categorical variable by assigning 2 or several categorical values if this results in a better model fit. Exploratory variables were entered using a stepwise regression algorithm using the following criteria: Variables must be significant at the 0.25 level to be entered into the model and significant at 0.15 level to remain in the model. For the final model, point estimates of hazard ratios are provided along with 95% CIs.

針對在篩選時WBC<10000μL之患者執行亞組分析。利用軟體 GraphPad Prism 5以及所分析之每一患者之原始數據實施額外亞組分析。 Subgroup analysis was performed for patients with WBC <10000 μL at screening. use software Additional subgroup analyzes were performed with GraphPad Prism 5 and the raw data for each patient analyzed.

效能結果：Performance results:

不探索其他共變量之關於無進展存活(PFS)之Cox比例危險模型分析指示A組(利用CBP501之組)之危險高於B組(HR=1.20[0.88,1.65])，但其在統計學上不顯著(P=0.25)。探索其他共變量之相同模型亦指示A組之危險高於B組(HR=1.21[0.85,1.73])，但其在統計學上不顯著(P=0.30)。 Cox proportional hazards model analysis for progression-free survival (PFS) without exploring other covariates indicated that group A (group using CBP501) had a higher risk than group B (HR=1.20[0.88,1.65]), but it was statistically Not significant (P=0.25). The same model exploring other covariates also indicated a higher risk in Group A than in Group B (HR=1.21 [0.85,1.73]), but it was not statistically significant (P=0.30).

對於在篩選時WBC<10000/μL之患者，不探索其他共變量之Cox比例危險模型分析指示A組之危險高於B組(HR=1.04[0.73,1.49])，但其在統計學上不顯著(P=0.81)。探索其他共變量之相同模型亦指示A組之危險高於B組(HR=1.06[0.71,1.59])，但其在統計學上不顯著(P=0.78)。 For patients with WBC<10000/μL at screening, Cox proportional hazards model analysis without exploring other covariates indicated that the risk was higher in group A than in group B (HR=1.04[0.73,1.49]), but it was not statistically significant. Significantly (P=0.81). The same model exploring other covariates also indicated a higher risk in Group A than in Group B (HR=1.06[0.71,1.59]), but it was not statistically significant (P=0.78).

應注意當在兩種分析(表A、表B)中，分析限於在篩選時WBC<10000/μL之患者時，A組中PFS之危險比有所改良。 It should be noted that the hazard ratio for PFS in arm A improved when the analysis was restricted to patients with WBC<10000/μL at screening in both analyzes (Table A, Table B).

在所有經治療群體上關於整體存活(OS)之Cox比例危險模型分析Cox proportional hazards model analysis for overall survival (OS) on all treated populations

在不探索及探索其他共變量之情形下，A組之危險均低於B組 (HR=0.96及0.77)。該差異在統計學上不顯著(P=0.82及0.25)。 In the case of not exploring and exploring other covariates, the risk of group A is lower than that of group B (HR=0.96 and 0.77). This difference was not statistically significant (P=0.82 and 0.25).

在篩選時WBC<10000/μL之患者上關於OS之Cox比例危險模型分析Cox proportional hazards model analysis for OS in patients with WBC<10000/μL at screening

對於在經治療群體中在篩選時WBC<10000/μL之患者上之OS，在不探索及探索其他共變量之情況下，A組之危險低於B組(HR=0.80及0.69)；該差異在統計上不顯著(P=0.32及0.16)。 For OS on patients with WBC<10000/μL at screening in the treated population, the risk was lower in Arm A than in Arm B (HR=0.80 and 0.69), without probing and exploring other covariates; this difference Statistically not significant (P=0.32 and 0.16).

應注意在所有分析(表C、表D)中當分析限於在篩選時WBC<10000/μL之患者時，A組中OS之危險比有所改良。 It should be noted that in all analyzes (Table C, Table D) the hazard ratios for OS improved in arm A when the analysis was restricted to patients with WBC<10000/μL at screening.

圖14顯示在所有經治療患者中關於篩選(基線)時之WBC之卡普蘭-邁耶存活曲線、中值OS及危險比。危險比隨截止含量降低而改良且在WBC 8000/μl下之峰值作為截止含量。 Figure 14 shows Kaplan-Meier survival curves, median OS and hazard ratios for WBC at screening (baseline) in all treated patients. Hazard ratios improved with decreasing cut-off levels and the peak at WBC 8000/μl was taken as the cut-off level.

參考圖17，在利用Hetasep(Stemcell technol.)去除紅血球之後，利用EasySep富嗜中性球套組(Stemcell technol.)自人類末梢血純化嗜中性球。將經純化嗜中性球(1×10⁶個細胞/孔，24孔板)與或不與1μM CBP501一起培養15分鐘(藉由添加EDTA終止反應)，且與1nM PMA、3μM或10μM A23187或100ng/ml或1000ng/ml LPS一起再培養4小時。將各孔洗滌兩次並與DNase一起培育15min並收集上清液，與彈性蛋白酶受質一起培育2小時，且然後加以分析以檢測彈性蛋白酶活性。 Referring to FIG. 17 , after red blood cells were removed using Hetasep (Stemcell technol.), neutrophils were purified from human peripheral blood using EasySep neutrophil-enriched kit (Stemcell technol.). Purified neutrophils (1×10 ⁶ cells/well, 24-well plate) were incubated with or without 1 μM CBP501 for 15 minutes (the reaction was terminated by adding EDTA), and mixed with 1 nM PMA, 3 μM or 10 μM A23187 or 100ng/ml or 1000ng/ml LPS were incubated for another 4 hours. Wells were washed twice and incubated with DNase for 15 min and the supernatant collected, incubated with elastase substrate for 2 hours, and then analyzed for elastase activity.

參考圖18，在注射苯海拉明之前的30分鐘利用或不用2.5ug/ml、5ug/ml或10ug/ml LPS靜脈內注射8週齡之C57BL/6小鼠。在注射苯海拉明之後的30分鐘注射CBP501(7.5mg/kg)。利用ELISA對自在注射CBP501或模擬物後3小時抽取之血液分離之血漿中之凝血酶/抗凝血酶複合物進行定量。該等數據係自四隻動物在每一條件下獲得的。 Referring to FIG. 18 , 8-week-old C57BL/6 mice were injected intravenously with or without 2.5ug/ml, 5ug/ml or 10ug/ml LPS 30 minutes before diphenhydramine injection. CBP501 (7.5 mg/kg) was injected 30 minutes after the injection of diphenhydramine. Thrombin/antithrombin complexes were quantified by ELISA in plasma isolated from blood drawn 3 hours after injection of CBP501 or mock. The data were obtained from four animals per condition.

參考圖19，藉由用0.32μM PMA刺激人類末梢血單核細胞並在PMA刺激48小時後去除所有懸浮細胞而獲得巨噬細胞。與50ng/ml IFN-γ、10ng/ml LPS一起進一步培育細胞以獲得M1表型，且與20ng/ml IL-4一起培育以獲得M2巨噬細胞。兩種治療均利用或不利用CBP501。藉由使用螢光標記之珠粒及流式細胞術監測吞噬活性。 Referring to FIG. 19 , macrophages were obtained by stimulating human peripheral blood mononuclear cells with 0.32 μM PMA and removing all suspended cells 48 hours after PMA stimulation. Cells were further incubated with 50 ng/ml IFN-γ, 10 ng/ml LPS to obtain the M1 phenotype, and 20 ng/ml IL-4 to obtain M2 macrophages. Both treatments were with or without CBP501. Phagocytic activity was monitored by using fluorescently labeled beads and flow cytometry.

參考圖20，將巨噬細胞系RAW264.7與或不與0.1μM或1μM CBP501一起培育3至6小時，且然後與或不與10ng/ml或1000ng/ml LPS再培育4小時。藉由ELISA量測所釋放之TNF。 Referring to FIG. 20 , the macrophage cell line RAW264.7 was incubated with or without 0.1 μM or 1 μM CBP501 for 3 to 6 hours, and then with or without 10 ng/ml or 1000 ng/ml LPS for another 4 hours. Released TNF was measured by ELISA.

CBP501顯示在臨床前(Sha,S.等人Mol.Cancer Ther.6：147(2007))及I期臨床研究(Shapiro,G.I.等人Clin.Cancer Res.(2011))中作為有效抗腫瘤劑之潛能。CBP501可在兩種作用機制下操作，該等作用機制係例如經由細胞週期G2查核點廢除(Sha,S.等人Mol.Cancer Ther.6：147(2007))及腫瘤細胞中之鉑濃度或藉助攜鈣蛋白抑制(Mine,N.等人Mol.Cancer Ther.10：1929(2011))。 CBP501 was shown as an effective antitumor agent in preclinical (Sha, S. et al. Mol. Cancer Ther. 6:147 (2007)) and phase I clinical studies (Shapiro, G.I. et al. Clin. Cancer Res. (2011)) potential. CBP501 can operate under two mechanisms of action, e.g., via abrogation of the cell cycle G2 checkpoint (Sha, S. et al. Mol. Cancer Ther. 6:147 (2007)) and platinum concentration in tumor cells or Inhibition by calpain (Mine, N. et al. Mol. Cancer Ther. 10:1929 (2011)).

如本文所證實，藉助非鱗狀非小細胞肺癌患者之II期臨床研究之患者群體上之亞組分析意外地發現，在篩選時具有高白血球計數(WBC)之患者群組與具有正常或低WBC之患者群組之間之存活的差異具有統計顯著性(p<0.0001)。 As demonstrated herein, patients with a phase II clinical study of patients with non-squamous non-small cell lung cancer Subgroup analysis across populations unexpectedly found a statistically significant (p<0.0001 ) difference in survival between the cohort of patients with high white blood count (WBC) at screening and those with normal or low WBC.

另外，如本文結果指示，當患者因由巨噬細胞引起NET之清除率/吞噬作用而發炎時，CBP501除直接作用於腫瘤細胞以外亦可藉由抑制攜鈣蛋白及增加嗜中性球細胞外陷阱(NET)而作用於腫瘤微環境(例如巨噬細胞)。此可增加具有深層靜脈血栓形成(DVT)及轉移之機會，且因此潛在地不利地影響患者存活。反之，抑制患者之M2型巨噬細胞可防止巨噬細胞對腫瘤生長、血管生成、轉移及腫瘤免疫逃避之積極作用，所有彼等皆促進腫瘤轉移，從而可縮短患者之存活。 In addition, as indicated by the results herein, when a patient is inflamed due to clearance/phagocytosis of NETs by macrophages, CBP501, in addition to directly acting on tumor cells, also acts by inhibiting calciproteins and increasing neutrophil extracellular traps. (NET) to act on the tumor microenvironment (such as macrophages). This can increase the chance of having deep vein thrombosis (DVT) and metastasis, and thus potentially adversely affects patient survival. Conversely, inhibition of M2-type macrophages in a patient prevents the positive effects of macrophages on tumor growth, angiogenesis, metastasis, and tumor immune evasion, all of which promote tumor metastasis, thereby shortening patient survival.

與此觀察一致，已證實藉由活體外CBP501治療增加之經活化嗜中性球之NET形成及LPS刺激之小鼠中增加之凝血酶/抗凝血酶複合物形成。亦已指示藉由CBP501可抑制M1型及M2型巨噬細胞之細胞介素分泌及吞噬作用。 Consistent with this observation, increased NET formation of activated neutrophils by CBP501 treatment in vitro and increased thrombin/antithrombin complex formation in LPS-stimulated mice were demonstrated. It has also been shown that cytokine secretion and phagocytosis of M1 and M2 macrophages can be inhibited by CBP501.

儘管臨床研究已指示CBP501係藉由增強順鉑對抗腫瘤細胞之細胞毒性起作用，但本文結果證實藉由對非鱗狀NSCLC患者之II期研究進行之亞組分析之意外發現，指示在臨床研究之篩選時具有高白血球計數(WBC)之患者群組存活較短，且具有正常或低WBC之其他患者群組因應利用CBP501之方案而存活較長，且該差異高度具有統計顯著性，且藉由對數秩(Mantel-Cox)測試的在關於整體存活之卡普蘭-邁耶曲線上計算之p值小於0.0001。 Although clinical studies have indicated that CBP501 acts by enhancing the cytotoxicity of cisplatin against tumor cells, the results herein confirm unexpected findings by a subgroup analysis of a phase II study of patients with non-squamous NSCLC, indicating that in clinical studies The patient cohort with high white blood cell count (WBC) at screening had shorter survival, and other patient cohorts with normal or low WBC survived longer in response to regimens utilizing CBP501, and this difference was highly statistically significant, and by The p-value calculated by the log-rank (Mantel-Cox) test on the Kaplan-Meier curve for overall survival was less than 0.0001.

因此意外地發現在治療前具有正常或低WBC之患者得益於CBP501治療，而具有高WBC之患者可能受到該相同治療的不利影響。CBP501對攜鈣蛋白之抑制活性表明對各種微環境細胞(例如巨噬細胞、白血球及淋巴球)之效應可抑制或調節該等細胞之活性，此可能由於(例如)藉由僅抑制巨噬細胞(若CBP501抑制M1型巨噬細胞，則其可不利地影響患者存活，且若CBP501抑制M2巨噬細胞，則其可延長患者存活)而促進雙向結果。 It was thus surprising to find that patients with normal or low WBC prior to treatment benefited from CBP501 treatment, whereas patients with high WBC could be adversely affected by this same treatment. CBP501 pair The inhibitory activity of calpains suggests that effects on various microenvironmental cells such as macrophages, leukocytes and lymphocytes may inhibit or modulate the activity of these cells, which may be due, for example, by inhibiting only macrophages (if CBP501 inhibits M1 macrophages, which can adversely affect patient survival, and if CBP501 inhibits M2 macrophages, it can prolong patient survival), promoting a bidirectional outcome.

已報導攜鈣蛋白抑制劑可抑制巨噬細胞(Horwitz,S.B.等人J.Cell Biol.91：798(1981)；Takenawa,T.等人Biochem J.15：208(1982)；Westra,J.等人BMC Musculoskelet.Disord.30：11(2010))、白血球(Naccache,P.H.等人Biochem.Biophys.Res.Commun.97(1)：62(1980)；Takeshige,K.等人Biochem.Biophys.Res.Commun.99(2)：484(1981)；Jones,H.P.等人Biochem Biophys.Acta.714(1)：152(1982)；Jones,H.P.等人Methods Enzymol.015：389(1984)；Verploegen,S.等人Eur.J.Biochem.269(18)4625(2002))及淋巴球(Salisbury,J.L.等人Nature 12：294(1981)；Boubali,S.等人Mol.Immunol.52(2)：51(2012))之多個功能。具有高WBC之患者將傾向於具有M1巨噬細胞(此乃因其為促發炎類型)且具有正常或低WBC且具有腫瘤之患者將傾向於具有M2巨噬細胞(Hao,N.等人Clin.Dev.Immunol.(2012))。另外，已知具有高WBC之患者更易於具有深層靜脈血栓形成(DVT)(Pabinger,I.等人，Blood 122：12(2013)；Blix,K.等人，PLOS One 4：8(2013)；Wang,T.F.等人，Thromb.Res.133(1)：25(2014))，此係顯著數量癌症患者之死亡原因。彼等患者傾向於具有更多NET，且NET促進腫瘤轉移(Cools-Lartigue,J.等人J.Clin.Invest.(2013))，此係許多癌症患者(包括患有肺癌之彼等)之短預後之一個原因。 Calpain inhibitors have been reported to inhibit macrophages (Horwitz, S.B. et al. J. Cell Biol. 91: 798 (1981); Takenawa, T. et al. Biochem J. 15: 208 (1982); Westra, J. et al. BMC Musculoskelet. Disord. 30: 11 (2010)), leukocytes (Naccache, P.H. et al. Biochem. Biophys. Res. Commun. 97(1): 62 (1980); Takeshige, K. et al. Biochem. Biophys. Res.Commun.99(2):484(1981); Jones, H.P. et al. Biochem Biophys.Acta.714(1):152(1982); Jones, H.P. et al. Methods Enzymol.015:389(1984); Verploegen , S. et al. Eur.J.Biochem.269 (18) 4625 (2002)) and lymphocytes (Salisbury, J.L. et al. Nature 12: 294 (1981); Boubali, S. et al. Mol. Immunol.52 (2 ): Multiple functions of 51(2012)). Patients with high WBC will tend to have M1 macrophages (because they are the pro-inflammatory type) and patients with normal or low WBC and tumors will tend to have M2 macrophages (Hao, N. et al. Clin . Dev. Immunol. (2012)). In addition, patients with high WBC are known to be more prone to deep vein thrombosis (DVT) (Pabinger, I. et al., Blood 122:12 (2013); Blix, K. et al., PLOS One 4:8 (2013) ; Wang, T.F. et al., Thromb. Res. 133(1):25(2014)), which is the cause of death in a significant number of cancer patients. These patients tend to have more NETs, and NETs promote tumor metastasis (Cools-Lartigue, J. et al. J. Clin. Invest. (2013)), which is the case for many cancer patients, including those with lung cancer. One reason for the short prognosis.

在此實例中，當在所有經治療群體中分析存活益處時，自將CBP501添加至標準方案(培美曲塞加順鉑)未檢測到統計顯著之存活益處。然而，藉由亞組分析意外地鑑別出，添加CBP501可為在針對臨床試驗篩選時顯示正常或低白血球(WBC)計數之人群提供益處。WBC之正常值因地點及國家而異。正常上限WBC可為8000/μl至11000/μl。 In this example, no statistically significant survival benefit was detected from the addition of CBP501 to the standard regimen (pemetrexed plus cisplatin) when the survival benefit was analyzed across all treated populations. However, It was unexpectedly identified by subgroup analysis that the addition of CBP501 could provide benefit to those who showed normal or low white blood cell (WBC) counts when screened for clinical trials. Normal values of WBC vary by location and country. Upper normal WBC can be 8000/μl to 11000/μl.

令人驚奇地，具有正常範圍之WBC之患者可得益於CBP501，且在篩選時具有高WBC之彼等較經順鉑及培美曲塞治療之患者更差，但在與對照組順鉑及培美曲塞治療之群體比較時，兩個差異在統計學上均不顯著。 Surprisingly, patients with normal-range WBC benefited from CBP501, and those with high WBC at screening performed worse than cisplatin- and pemetrexed-treated patients, but compared with control cisplatin Neither difference was statistically significant when compared with the pemetrexed-treated group.

儘管並不明瞭CBP501之此潛在雙向作用之確切原因，但CBP501對攜鈣蛋白之抑制作用指示各種微環境細胞(例如巨噬細胞、白血球及淋巴球)中攜鈣蛋白之抑制抑制或調節該等細胞之活性，此由於以下原因而促進雙向結果：例如，若CBP501抑制M1型巨噬細胞，則其可因抑制巨噬細胞之抗腫瘤活性及/或抑制NET之清除率而不利地影響患者存活，乃因NET促進血栓形成及轉移，且若CBP501抑制促腫瘤M2巨噬細胞，則其可延長患者存活。另外，已知順鉑可使巨噬細胞自M1型變為M2型(Dijkgraaf,E.M.等人Cancer Res.15：73(8)：2480(2013))，且已知化學療法本身可促進腫瘤轉移(Haas,M.J.SciBX 1-3(2011))，因此在化學療法開始時存在CBP501可對腫瘤轉移具有顯著影響。已知攜鈣蛋白抑制劑能夠抑制巨噬細胞(Horwitz,S.B.等人J.Cell Biol.91：798(1981)；Takenawa,T.等人Biochem J.15：208(1982)；Westra,J.等人BMC Musculoskelet.Disord.30：11(2010))、白血球(Naccache,P.H.等人Biochem.Biophys.Res.Commun.97(1)：62(1980)；Takeshige,K.等人Biochem.Biophys.Res.Commun.99(2)：484(1981)；Jones,H.P.等人Biochem Biophys.Acta.714(1)：152(1982)；Jones,H.P.等人Methods Enzymol.015：389(1984)；Verploegen,S.等人Eur.J.Biochem.269(18)4625(2002))及淋巴球(Salisbury,J.L.等人Nature 12：294(1981)；Boubali,S.等人Mol.Immunol.52(2)：51(2012))之多個功能。具有高WBC之患者將傾向於具有更多M1巨噬細胞(此乃因其係促發炎的)，且具有正常或低WBC且具有腫瘤之患者將傾向於具有M2巨噬細胞(Hao,N.等人Clin.Dev.Immunol.(2012))。另外，具有高WBC之患者將傾向於具有更多NET。若CBP501阻止NET之吞噬作用，則患者具有DVT及轉移之風險可能更大，此二者均可縮短存活時間。 Although the exact reason for this potential bi-directional effect of CBP501 is not understood, the inhibition of calciportin by CBP501 indicates that inhibition of calciportin in various microenvironmental cells (such as macrophages, leukocytes, and lymphocytes) inhibits or regulates these Cellular activity, which contributes to a bidirectional outcome because, for example, if CBP501 inhibits M1 macrophages, it can adversely affect patient survival by inhibiting the antitumor activity of the macrophages and/or inhibiting the clearance of NETs , because NETs promote thrombus formation and metastasis, and if CBP501 inhibits tumor-promoting M2 macrophages, it prolongs patient survival. In addition, cisplatin is known to change macrophages from M1 to M2 type (Dijkgraaf, E.M. et al. Cancer Res. 15:73(8):2480 (2013)), and chemotherapy itself is known to promote tumor metastasis (Haas, M.J. SciBX 1-3 (2011)), therefore the presence of CBP501 at the initiation of chemotherapy can have a significant impact on tumor metastasis. Calpain inhibitors are known to inhibit macrophages (Horwitz, S.B. et al. J. Cell Biol. 91: 798 (1981); Takenawa, T. et al. Biochem J. 15: 208 (1982); Westra, J. et al. BMC Musculoskelet. Disord. 30: 11 (2010)), leukocytes (Naccache, P.H. et al. Biochem. Biophys. Res. Commun. 97(1): 62 (1980); Takeshige, K. et al. Biochem. Biophys. Res.Commun.99(2):484(1981); Jones, H.P. et al. Biochem Biophys.Acta.714(1):152(1982); Jones, H.P. et al. Methods Enzymol.015:389(1984); Verploegen , S. et al. Eur.J.Biochem.269 (18) 4625 (2002)) and lymphocytes (Salisbury, J.L. et al. Human Nature 12: 294 (1981); Boubali, S. et al. Mol. Immunol. 52(2): 51 (2012)) multiple functions. Patients with high WBC will tend to have more M1 macrophages because they are pro-inflammatory, and patients with normal or low WBC and tumors will tend to have M2 macrophages (Hao, N. et al. Clin. Dev. Immunol. (2012)). Additionally, patients with high WBC will tend to have more NETs. If CBP501 prevents phagocytosis of NETs, patients may be at greater risk of DVT and metastasis, both of which can shorten survival time.

或者，攜鈣蛋白參與白血球之正常功能(Horwitz,S.B.等人J.Cell Biol.91：798(1981)；Takenawa,T.等人Biochem J.15：208(1982)；Westra,J.等人BMC Musculoskelet.Disord.30：11(2010)；Naccache,P.H.等人Biochem.Biophys.Res.Commun.97(1)：62(1980)；Takeshige,K.等人Biochem.Biophys.Res.Commun.99(2)：484(1981)；Jones,H.P.等人Biochem Biophys.Acta.714(1)：152(1982)；Jones,H.P.等人Methods Enzymol.015：389(1984)；Verploegen,S.等人Eur.J.Biochem.269(18)4625(2002)；Salisbury,J.L.等人Nature 12：294(1981)；Boubali,S.等人Mol.Immunol.52(2)：51(2012)；Hao,N.等人Clin.Dev.Immunol.(2012))。當非常需要該等WBC時(例如當患者罹患使白血球計數增加之活動性感染時之情形)，CBP501可因可危害WBC之功能而干擾及抵消其對整體存活之有益活性。 Alternatively, calpain is involved in the normal function of leukocytes (Horwitz, S.B. et al. J. Cell Biol. 91: 798 (1981); Takenawa, T. et al. Biochem J. 15: 208 (1982); Westra, J. et al. BMC Musculoskelet. Disord. 30: 11 (2010); Naccache, P.H. et al. Biochem. Biophys. Res. Commun. 97(1): 62 (1980); Takeshige, K. et al. Biochem. Biophys. Res. Commun. 99 (2): 484 (1981); Jones, H.P. et al. Biochem Biophys. Acta. 714(1): 152 (1982); Jones, H.P. et al. Methods Enzymol. 015: 389 (1984); Verploegen, S. et al. Eur.J.Biochem.269(18)4625(2002); Salisbury, J.L. et al. Nature 12:294 (1981); Boubali, S. et al. Mol.Immunol.52(2):51(2012); Hao, N. et al. Clin. Dev. Immunol. (2012)). When these WBCs are in great need, such as is the case when a patient suffers from an active infection that raises white blood cell counts, CBP501 can interfere and counteract their beneficial activity on overall survival by compromising WBC function.

攜鈣蛋白抑制亦可藉由作用於淋巴球影響抗癌免疫性，因為已表明攜鈣蛋白可誘導T細胞無反應性(Boubali,S.等人Mol.Immunol.52(2)：51(2012))。 Calpain inhibition may also affect anticancer immunity by acting on lymphocytes, as calpain has been shown to induce T cell anergy (Boubali, S. et al. Mol. Immunol. 52(2):51(2012 )).

治療前或基線WBC計數已經指示為經基於鉑之療法治療之NSCLC患者之預後因素(Teramukai,S.等人Eur.J.Cancer(45(11：1950(2009)；Kim, J.W.等人cancer Res.Trest.45：4)：325(2013))。CBP501可藉由調節順鉑之活性而增強此效應。由於每名患者將在治療前獲得WBC計數之實驗室分析作為普遍批准之標準程序，故可基於WBC計數選擇患者。 Pre-treatment or baseline WBC counts have been indicated as prognostic factors in NSCLC patients treated with platinum-based therapy (Teramukai, S. et al. Eur. J. Cancer (45 (11:1950 (2009); Kim, J.W. et al. Cancer Res. Trest. 45:4):325 (2013)). CBP501 can enhance this effect by modulating the activity of cisplatin. Patients can be selected based on WBC counts as each patient will have laboratory analysis of WBC counts obtained prior to treatment as a generally accepted standard procedure.

CBP501對攜鈣蛋白之抑制亦可獨立於鉑濃度而直接抑制腫瘤遷移及轉移，因為攜鈣蛋白已顯示在遷移中起重要作用(Wang,H.等人Nat.Commun.4：1354(2013))。 Inhibition of calpains by CBP501 may also directly inhibit tumor migration and metastasis independent of platinum concentration, as calpains have been shown to play an important role in migration (Wang, H. et al. Nat. Commun. 4: 1354 (2013) ).

實例7Example 7

此實例包括用於治療癌症之CBP501組合之闡述。 This example includes a description of CBP501 combinations for the treatment of cancer.

CBP501係已針對患有惡性胸膜間皮瘤及非小細胞肺癌(NSCLC)之患者完成兩項II期臨床試驗之抗癌症藥物。CBP501經闡述為獨特G2查核點靶向劑(1)且為順鉑(CDDP)攝取之增強劑(2)。 CBP501 is an anti-cancer drug that has completed two phase II clinical trials for patients with malignant pleural mesothelioma and non-small cell lung cancer (NSCLC). CBP501 has been described as a unique G2 checkpoint targeting agent (1) and as an enhancer of cisplatin (CDDP) uptake (2).

CDDP係已知之最有效化學治療劑之一，其顯示抵抗眾多種實體腫瘤之臨床活性。據信主要作用模式為共價結合至DNA而引起特徵性生物效應，此最終活化細胞凋亡程式(3)。CDDP之抗腫瘤活性可能不限於其抑制腫瘤細胞之有絲分裂之能力，且亦包括重要的免疫調節效應。De Biasi等人概述了CDDP誘導之抗腫瘤免疫調節之四種主要機制，該等機制為1)I類MHC上調，2)招募及增殖效應細胞，3)上調細胞毒性效應物之溶解活性，及4)藉由系統地檢查相關臨床前文獻，下調免疫抑制微環境(4)。 CDDP is one of the most potent chemotherapeutic agents known, showing clinical activity against a wide variety of solid tumors. The primary mode of action is believed to be covalent binding to DNA causing characteristic biological effects, which ultimately activate the apoptotic program (3). The antitumor activity of CDDP may not be limited to its ability to inhibit the mitosis of tumor cells, but also includes important immunomodulatory effects. De Biasi et al. outline four major mechanisms of CDDP-induced antitumor immune modulation, which are 1) upregulation of MHC class I, 2) recruitment and proliferation of effector cells, 3) upregulation of lytic activity of cytotoxic effectors, and 4) Downregulation of the immunosuppressive microenvironment by systematically examining relevant preclinical literature (4).

免疫原性細胞死亡(ICD)係功能上獨特之細胞凋亡形式，其可使免疫活性宿主足以調用抵抗死亡細胞相關性抗原之適應性免疫反應。若干藥物包括各種化學治療劑(例如多柔比星、泛艾黴素、伊達比星、米托蒽醌、博來黴素、硼替佐米(bortezomib)、環磷醯胺及奧沙利鉑)，其為ICD誘導物(5)。ICD需要三種特徵標誌，包括細胞表面鈣網蛋白暴露及ATP釋放及高遷移率族蛋白B1(high-mobility group box 1 protein，HMGB1)(6)。據報導CDDP為相對較差之ICD誘導物(7)。最近，Aranda等人報導CDDP與維生素B6前體吡哆醇之組合引起ICD(包括ER應力反應及細胞表面鈣網蛋白暴露)(8)。吡哆醇之一個功能係以PDXK依賴性方式加強CDDP之細胞內累積(9)。儘管吡哆醇與CBP501之間之作用機制看似不同，但CDDP累積之增加係類似的。 Immunogenic cell death (ICD) is a functionally unique form of apoptosis that enables an immunocompetent host to mount an adaptive immune response against antigens associated with dying cells. Several drugs including various chemotherapeutic agents (eg, doxorubicin, pan-rubycin, idarubicin, mitoxantrone, bleomycin, bortezomib, cyclophosphamide, and oxaliplatin) , which is an ICD inducer (5). ICD requires three hallmarks, including cell surface calreticulin exposure and ATP release and high Mobility group box 1 protein (HMGB1) (6). CDDP has been reported to be a relatively poor inducer of ICD (7). Recently, Aranda et al. reported that the combination of CDDP and the vitamin B6 precursor pyridoxine caused ICD (including ER stress response and cell surface calreticulin exposure) (8). One function of pyridoxine is to potentiate the intracellular accumulation of CDDP in a PDXK-dependent manner (9). Although the mechanisms of action between pyridoxine and CBP501 appear to be different, the increase in CDDP accumulation is similar.

癌細胞藉由各種方式逃避免疫監督，包括使用免疫查核點，其藉由在T細胞上結合抑制性受體(例如CTLA-4或PD-1)而防止細胞毒性T細胞攻擊腫瘤細胞(10)。對免疫查核點抑制劑(例如抗PD1抗體及抗CTLA4抗體)之反應可能令人驚歎；然而，許多患者對該等抗體沒有反應。 Cancer cells evade immune surveillance in various ways, including the use of immune checkpoints, which prevent cytotoxic T cells from attacking tumor cells by binding inhibitory receptors (such as CTLA-4 or PD-1) on T cells (10) . Responses to immune checkpoint inhibitors, such as anti-PD1 antibodies and anti-CTLA4 antibodies, can be surprising; however, many patients do not respond to these antibodies.

如本文所揭示，CBP501加強CDDP誘導之ICD指示物之出現。CBP501亦與抗PD-1抗體組合在免疫活性小鼠模型中促進抗腫瘤效應。 As disclosed herein, CBP501 potentiates the appearance of CDDP-induced ICD indicators. CBP501 also promotes anti-tumor effects in combination with anti-PD-1 antibodies in immunocompetent mouse models.

實例8Example 8

此實例包括材料及方法之闡述。 This example includes a description of the Materials and methods.

細胞週期分析Cell cycle analysis

為進行細胞週期分析，利用克裡尚氏緩衝液(0.1%檸檬酸鈉，50Ag/mL碘化丙啶，20Ag/mL RNase A,0.5% NP40)對藉由胰蛋白酶/EDTA(Gibco)收集之細胞進行染色，隨後使用FACSCalibur(Becton Dickinson)實施流式細胞術。 For cell cycle analysis, cells collected by trypsin/EDTA (Gibco) were digested with Chrysanthemum buffer (0.1% sodium citrate, 50 Ag/mL propidium iodide, 20 Ag/mL RNase A, 0.5% NP40). Cells were stained followed by flow cytometry using a FACSCalibur (Becton Dickinson).

Analysis of markers of immunogenic cell death

利用CDDP(10μM或20μM)及0.5μM CBP501將CT26WT(CRL-2638,ATCC)(CBP501敏感性細胞品系)活體外處理0.75小時。在更換為新鮮的不含藥物之培養基後，將細胞培育24小時、48小時或72小時用於磷酸化eIF2-α之免疫印漬分析，用於藉由FACS進行鈣網蛋白分析，或用於HMGB1 ELISA。 CT26WT (CRL-2638, ATCC) (CBP501-sensitive cell line) was treated with CDDP (10 μM or 20 μM) and 0.5 μM CBP501 for 0.75 hours in vitro. After replacing with fresh drug-free medium, cells were incubated for 24 hours, 48 hours or 72 hours for Immunoblot analysis on phosphorylated eIF2-α for calreticulin analysis by FACS, or for HMGB1 ELISA.

western blot

藉由在4℃下與緩衝液[NaCl(100mM),Tris-HCl(50mM,pH 8.0),DTT(1mM),NP40(0.5w/v%)，含有PhosSTOP磷酸酶抑制劑混合劑及完全蛋白酶抑制劑混合劑錠劑(Roche Life Science)，每一者均稀釋至製造商之指定濃度]一起培育30分鐘來溶解細胞並進行離心(15,000rpm,4℃,20分鐘)。對上清液(全細胞溶解物)實施電泳(SDS-PAGE)，並藉由電印漬將其自凝膠轉移至聚偏氟乙烯(PVDF)膜(Millipore)上。在室溫下利用1%於T-TBS中之Block Ace阻斷1小時後，在4℃下將PVDF膜與一級抗體一起培育過夜。第二天，用T-TBS洗滌膜並在室溫下與二級抗體-HRP偶聯物一起培育1小時。在利用T-TBS洗滌PVDF膜後，利用Immobilon Western HRP檢測受質(Millipore)將蛋白質可視化。利用Luminescent Image Analyzer LAS-4000系統(Fujifilm)記錄影像，並利用Multi Gauge軟體(Fujifilm)對譜帶強度進行定量。 By mixing with buffer [NaCl (100mM), Tris-HCl (50mM, pH 8.0), DTT (1mM), NP40 (0.5w/v%) at 4°C, containing PhosSTOP phosphatase inhibitor mixture and complete protease Inhibitor Cocktail Tablets (Roche Life Science), each diluted to the concentration specified by the manufacturer] were incubated together for 30 minutes to lyse the cells and centrifuged (15,000 rpm, 4°C, 20 minutes). The supernatant (whole cell lysate) was subjected to electrophoresis (SDS-PAGE) and transferred from the gel to a polyvinylidene fluoride (PVDF) membrane (Millipore) by electroblotting. After blocking with 1% Block Ace in T-TBS for 1 hour at room temperature, PVDF membranes were incubated with primary antibodies overnight at 4°C. The next day, membranes were washed with T-TBS and incubated with secondary antibody-HRP conjugates for 1 hr at room temperature. After washing the PVDF membrane with T-TBS, proteins were visualized using Immobilon Western HRP detection substrate (Millipore). Images were recorded using Luminescent Image Analyzer LAS-4000 system (Fujifilm), and band intensities were quantified using Multi Gauge software (Fujifilm).

細胞表面鈣網蛋白暴露之分析Analysis of cell surface calreticulin exposure

收集細胞並利用PBS洗滌，隨後利用含有0.25% PFA之PBS固定5分鐘。利用冰冷的PBS將經固定細胞洗滌兩次並在冰上用利用含有2% FBS(1/200)之PBS稀釋之抗鈣網蛋白抗體(abcam)處理30分鐘。用含有2% FBS洗滌液之PBS將一級抗體移除兩次，並在RT下用利用含有2% FBS(1/500)之PBS稀釋之alexa488偶聯之二級抗體處理30分鐘。用PBS將二級抗體移除兩次並在冰上利用碘化丙啶(1ug/ml)處理5分鐘以上，隨後使用FACSCalibur進行FACS分析。 Cells were harvested and washed with PBS, then fixed with PBS containing 0.25% PFA for 5 minutes. Fixed cells were washed twice with ice-cold PBS and treated with anti-calreticulin antibody (abcam) diluted in PBS containing 2% FBS (1/200) for 30 minutes on ice. Primary antibodies were removed twice with PBS containing 2% FBS washes and treated with alexa488-conjugated secondary antibodies diluted in PBS containing 2% FBS (1/500) for 30 min at RT. Secondary antibodies were removed twice with PBS and treated with propidium iodide (lug/ml) for 5+ minutes on ice, followed by FACS analysis using a FACSCalibur.

HMGB1 ELISA

藉由HMGB1 ELISA套組II(SINO-TEST CORPORATION,kanagawa,Japan)根據製造商之說明書對分泌至培養基之HMGB1進行定量。 HMGB1 secreted into the medium was quantified by HMGB1 ELISA Kit II (SINO-TEST CORPORATION, kanagawa, Japan) according to the manufacturer's instructions.

實例9Example 9

此實例包括使用單獨CBP501及其各種組合之活體內研究之闡述。 This example includes a description of in vivo studies using CBP501 alone and its various combinations.

所有動物研究皆根據由CanBas Co.Ltd之動物護養機構委員會批准之方案來執行。利用CT26WT之懸浮液(5×10⁵個細胞)在側腹對六週齡雌性Balb/c小鼠(Charles River Laboratories Japan Inc.)實施皮下接種。一週後，將小鼠分配至7個群組中(6隻小鼠/組)，並利用3種單獨抗癌劑或其不同組合之3個投藥週期進行治療[CDDP：5mg/kg×1/週，CBP501：7.5mg/kg×3/週，抗mPD1抗體(RMP1-14)：200ug×1/週]。在每一5%葡萄糖(CBP501之媒劑)或CBP501治療前的30min利用10mg/kg苯海拉明預治療小鼠。利用以下公式計算腫瘤體積(每週利用一對測徑器量測三次)：體積(mm³)=[寬度(mm)]²×長度(mm)/2。藉由繪製腫瘤大小之平均值+/- SE之圖形生成治療後之生長曲線。 All animal studies were performed according to protocols approved by the Institutional Animal Care Committee of CanBas Co. Ltd. Six-week-old female Balb/c mice (Charles River Laboratories Japan Inc.) were inoculated subcutaneously in the flank with a suspension of CT26WT (5×10 ⁵ cells). One week later, the mice were divided into 7 groups (6 mice/group) and treated with 3 administration cycles of 3 anticancer agents alone or in different combinations [CDDP: 5 mg/kg×1/ week, CBP501: 7.5mg/kg×3/week, anti-mPD1 antibody (RMP1-14): 200ug×1/week]. Mice were pre-treated with 10 mg/kg diphenhydramine 30 min before each 5% glucose (vehicle of CBP501) or CBP501 treatment. Tumor volumes (measured three times a week with a pair of calipers) were calculated using the following formula: Volume (mm ³ )=[Width (mm)] ² ×Length (mm)/2. Post-treatment growth curves were generated by graphing mean +/- SE of tumor size.

在CBP501敏感性人類癌細胞系中，與CBP501組合之CDDP較單獨CDDP可使CDDP之細胞內累積加強約大1.5倍至3倍。在CBP501敏感性鼠類細胞CT26WT中，CDDP與CBP501組合之治療在改變細胞週期期分佈方面較單獨CDDP強約2倍，此在許多情形下與細胞內鉑含量大致相關(圖21)。 In CBP501-sensitive human cancer cell lines, CDDP in combination with CBP501 enhanced the intracellular accumulation of CDDP approximately 1.5- to 3-fold greater than CDDP alone. In CBP501-sensitive murine CT26WT cells, treatment with CDDP in combination with CBP501 was approximately 2-fold more potent than CDDP alone in altering cell cycle phase distribution, which in many cases roughly correlated with intracellular platinum levels (Figure 21).

據報導eIF2-α中絲胺酸51之磷酸化(磷酸化eIF2-α)為ER應力之標記物，相信其係細胞表面鈣網蛋白暴露所必需的(11)。吾人分析CBP501抵抗CDDP誘導之磷酸化eIF2-α及細胞表面鈣網蛋白暴露之效應。 Phosphorylation of serine 51 in eIF2-α (phosphorylated eIF2-α) has been reported as a marker of ER stress and is believed to be required for cell surface calreticulin exposure (11). We analyzed the effect of CBP501 on CDDP-induced phosphorylated eIF2-α and cell surface calreticulin exposure.

CDDP治療以劑量依賴性方式上調eIF2-α之磷酸化，且CBP501可強化此(圖22)。在鈣網蛋白暴露於細胞表面中亦證實了CBP501之正面效應(圖23)。此外，CDDP與CBP501之組合所引起之HMGB1釋放較單獨CDDP約大2倍(圖24)。該等結果指示，CBP501可加強CDDP誘導之ICD。 CDDP treatment upregulated the phosphorylation of eIF2-α in a dose-dependent manner, and CBP501 could potentiate this (Figure 22). The positive effect of CBP501 was also confirmed in the exposure of calreticulin to the cell surface (Figure 23). Furthermore, the combination of CDDP and CBP501 caused approximately 2-fold greater release of HMGB1 than CDDP alone (Figure 24). These results indicate that CBP501 can potentiate CDDP-induced ICD.

基於CBP501之此新穎活性，亦使用具有s.c.CT26WT之BALB/c分析與活體內PD-1阻斷抗體組合之治療(圖25)。當腫瘤大小達到約100mm³時開始治療。 Based on this novel activity of CBP501, BALB/c assay with scCT26WT was also used in combination with in vivo PD-1 blocking antibody therapy ( FIG. 25 ). Treatment was initiated when the tumor size reached approximately 100 ^mm3 .

與媒劑治療之小鼠相比，CDDP治療之小鼠顯示腫瘤生長降低52.7%。與媒劑治療之小鼠相比，CBP501+CDDP顯示腫瘤生長另外降低63.1%。與媒劑治療之小鼠相比，單獨抗mPD-1抗體之治療顯示腫瘤生長略降低25.2%。然而，與媒劑治療之小鼠相比，抗mPD-1+CDDP或抗mPD-1+CDDP+CBP501之組合治療顯示腫瘤生長分別顯著降低69.3%及78.7%。 CDDP-treated mice showed a 52.7% reduction in tumor growth compared to vehicle-treated mice. CBP501+CDDP showed an additional 63.1% reduction in tumor growth compared to vehicle-treated mice. Treatment with anti-mPD-1 antibody alone showed a slight 25.2% reduction in tumor growth compared to vehicle-treated mice. However, combination treatment with anti-mPD-1+CDDP or anti-mPD-1+CDDP+CBP501 showed a significant reduction in tumor growth of 69.3% and 78.7%, respectively, compared to vehicle-treated mice.

References for Examples 7 to 9:

1. Sha等人，Cell cycle phenotype-based optimization of G2-abrogating peptides yields CBP501 with aunique mechanism of action at the G2 checkpoint.Mol Cancer Ther.2007；6：147-53。 1. Sha et al., Cell cycle phenotype-based optimization of G2-abrogating peptides yields CBP501 with aunique mechanism of action at the G2 checkpoint. Mol Cancer Ther. 2007; 6: 147-53.

2. Mine等人，CBP501-calmodulin binding contributes to sensitizing tumor cells to cisplatin and bleomycin.Mol Cancer Ther.2011；10：1929-38。 2. Mine et al., CBP501-calmodulin binding contributions to sensitizing tumor cells to cisplatin and bleomycin. Mol Cancer Ther. 2011; 10: 1929-38.

3. Siddik ZH. Cisplatin：mode of cytotoxic action and molecular basis of resistance.Oncogene.2003；22：7265-79。 3. Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene. 2003; 22: 7265-79.

4. De Biasi等人，Cisplatin-induced antitumor immunomodulation：a review of preclinical and clinical evidence.Clin Cancer Res. 2014；20：5384-91。 4. De Biasi et al., Cisplatin-induced antitumor immunomodulation: a review of preclinical and clinical evidence. Clin Cancer Res. 2014;20:5384-91.

5. Pol等人，Trial Watch：Immunogenic cell death inducers for anticancer chemotherapy.Oncoimmunology.2015；4：e1008866。 5. Pol et al., Trial Watch: Immunogenic cell death inducers for anticancer chemotherapy. Oncoimmunology. 2015; 4: e1008866.

6. Bianchi等人，Killing cancer cells,twice with one shot.Cell Death Differ.2014；21：1-2。 6. Bianchi et al., Killing cancer cells, twice with one shot. Cell Death Differ. 2014; 21: 1-2.

7. Tesniere等人，Immunogenic death of colon cancer cells treated with oxaliplatin.Oncogene.2010；29：482-91。 7. Tesniere et al., Immunogenic death of colon cancer cells treated with oxaliplatin. Oncogene. 2010; 29: 482-91.

8. Aranda等人，Immune-dependent antineoplastic effects of cisplatin plus pyridoxine in non-small-cell lung cancer.Oncogene.2015；34：3053-62。 8. Aranda et al., Immune-dependent antineoplastic effects of cisplatin plus pyridoxine in non-small-cell lung cancer. Oncogene. 2015; 34: 3053-62.

9. Galluzzi等人，Vitamin B6 metabolism influences the intracellular accumulation of cisplatin.Cell Cycle.2013；12：417-21。 9. Galluzzi et al., Vitamin B6 metabolism influences the intracellular accumulation of cisplatin. Cell Cycle. 2013; 12: 417-21.

10. Intlekofer AM及Thompson CB.At the bench：preclinical rationale for CTLA-4 and PD-1 blockade as cancer immunotherapy.J Leukoc Biol.2013；94：25-39。 10. Intlekofer AM and Thompson CB. At the bench: preclinical rationale for CTLA-4 and PD-1 blockade as cancer immunotherapy. J Leukoc Biol. 2013; 94: 25-39.

11. Panaretakis等人，Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death.EMBO J.2009；28：578-90。 11. Panaretakis et al., Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J. 2009;28:578-90.

實例10Example 10

此實例包括針對CBP501與順鉑之組合及抗PD1及CBP501與順鉑及抗PD-L1之組合之兩項異種移植物研究之闡述。 This example includes a description of two xenograft studies for the combination of CBP501 and cisplatin and anti-PD1 and CBP501 in combination with cisplatin and anti-PD-L1.

動物研究animal research

將CT26WT細胞(5×10⁵)皮下接種於8至9週齡雌性Balb/c小鼠之右側腹中。7天或8天後，基於腫瘤大小將小鼠隨機化，且在第1天開始組合療法。每週將腫瘤大小及體重量測兩次或三次。利用數位卡尺計算腫瘤大小，且利用以下公式計算體積：體積(mm³)=[寬度(mm)]²×長度(mm)/2。繪製腫瘤之相對大小對開始治療後之天數之圖形。 CT26WT cells (5×10 ⁵ ) were inoculated subcutaneously in the right flank of 8- to 9-week-old female Balb/c mice. After 7 or 8 days, mice were randomized based on tumor size, and combination therapy was started on day 1. Tumor size and body weight were measured twice or three times a week. Tumor size was calculated using a digital caliper, and volume was calculated using the following formula: Volume (mm ³ )=[Width (mm)] ² ×Length (mm)/2. The relative size of the tumors was plotted against the number of days after initiation of treatment.

CDDP與抗PD-1或抗PD-L1CDDP with anti-PD-1 or anti-PD-L1

在第1天及第8天以15分鐘間隔，腹膜內投與具有皮下CT26WT腫瘤之小鼠(n=6)苯海拉明(10mg/kg)，隨後靜脈內注射媒劑(鹽水)或CDDP(4mg/kg)或CDDP(4mg/kg)與CBP501(6mg/kg)之混合物。在第2天、第4天、第9天及第11天腹膜內投與包括抗PD-1(RMP1-14)及抗PD-L1(純系10F.9G2)之抗體(200μg/小鼠)或鹽水。 Diphenhydramine (10 mg/kg) was administered intraperitoneally to mice bearing subcutaneous CT26WT tumors (n=6) at 15-minute intervals on days 1 and 8, followed by intravenous injection of vehicle (saline) or CDDP (4mg/kg) or a mixture of CDDP (4mg/kg) and CBP501 (6mg/kg). Antibodies (200 μg/mouse) including anti-PD-1 (RMP1-14) and anti-PD-L1 (clone 10F.9G2) were administered intraperitoneally on days 2, 4, 9, and 11 or brine.

卡鉑與抗PD-L1Carboplatin and anti-PD-L1

在第1天及第8天以15分鐘間隔，腹膜內投與具有皮下CT26WT腫瘤之小鼠(n=9)苯海拉明(10mg/kg)，隨後靜脈內注射媒劑(鹽水)或卡鉑(50mg/kg)或卡鉑(50mg/kg)與CBP501(6mg/kg)之混合物。在第4天、第11天及第16天腹膜內投與四百微克抗PD-L1(純系10F.9G2)或鹽水。 Mice with subcutaneous CT26WT tumors (n=9) were administered diphenhydramine (10 mg/kg) intraperitoneally at 15-minute intervals on days 1 and 8, followed by intravenous injection of vehicle (saline) or carboxylate A mixture of platinum (50mg/kg) or carboplatin (50mg/kg) and CBP501 (6mg/kg). Four hundred micrograms of anti-PD-L1 (clone 10F.9G2) or saline were administered intraperitoneally on days 4, 11 and 16.

實例11Example 11

此實例包括腫瘤浸潤性淋巴球(TIL)之分析之闡述。 This example includes a description of the analysis of tumor infiltrating lymphocytes (TIL).

將CT26WT皮下接種至雌性Ba1b/c小鼠之左腹側中。7天至8天後，藉由腫瘤大小將小鼠隨機化至6個群組(3至4隻小鼠/組)，並利用媒劑、CDDP(4mg/kg i.v.)、Combo(CDDP 4mg/kg i.v.+CBP501 6mg/kg i.v.)、抗PD1(400ug/小鼠i.p.)、抗PD1加CDDP或抗PD-1加Combo進行治療。在第1天及第8天注射CDDP及CBP501，且在第2天、第5天及第8天注射抗PD1。然後在第11天自小鼠提取腫瘤，並利用三元酶混合物(膠原酶、玻尿酸酶及DNase)消化，以獲得單一細胞懸浮物。利用FcR阻斷劑處理細胞並利用用於多色流式細胞術分析之特定抗體進行染色，包括CD4、CD8、CD45、CD11b、Ly6G、Ly6C、F4/80、II類MHC及CD206。定量數據(圖32)顯示為平均值+SEM(n=3-4隻小鼠/組)。如與來自媒劑治療或PD-1治療之腫瘤之細胞相比，*：p<0.05，**：p<0.01，n.s.：不顯著(不成對之雙側司徒登氏t測試)。 CT26WT was inoculated subcutaneously into the left flank of female Ba1b/c mice. After 7 to 8 days, mice were randomized into 6 groups (3 to 4 mice/group) by tumor size, and treated with vehicle, CDDP (4 mg/kg i.v.), Combo (CDDP 4 mg/kg i.v. kg i.v.+CBP501 6mg/kg i.v.), anti-PD1 (400ug/mouse i.p.), anti-PD1 plus CDDP or anti-PD-1 plus Combo for treatment. CDDP and CBP501 were injected on days 1 and 8, and anti-PD1 was injected on days 2, 5 and 8. Tumors were then extracted from the mice on day 11 and digested with a ternary enzyme mix (collagenase, hyaluronidase and DNase) to obtain a single cell suspension. Treat cells with FcR blockers and use Stained with specific antibodies for multicolor flow cytometry analysis, including CD4, CD8, CD45, CD11b, Ly6G, Ly6C, F4/80, MHC class II and CD206. Quantitative data (Figure 32) are shown as mean + SEM (n=3-4 mice/group). *: p<0.05, **: p<0.01, n.s.: not significant (unpaired two-sided Studen's t-test) as compared to cells from vehicle-treated or PD-1-treated tumors.

上述數據證實CBP501在與CDDP組合時意欲增加CD8(=CD45+CD8+)(殺死腫瘤細胞之效應物T細胞群體)在腫瘤位點處之浸潤。當與CDDP及抗PD1抗體組合時，所達到值係統計顯著的。此結果與在小鼠異種移植物模型中觀察到之腫瘤生長抑制一致。 The data above demonstrate that CBP501, when combined with CDDP, intends to increase the infiltration of CD8 (=CD45+CD8+), the effector T cell population that kills tumor cells, at the tumor site. The values achieved were statistically significant when combined with CDDP and anti-PD1 antibody. This result is consistent with the tumor growth inhibition observed in the mouse xenograft model.

上述數據亦證實CBP501在與CDDP或CDDP加抗PD1抗體組合時可以統計顯著程度減少支持腫瘤生長及免疫逃逸之M2巨噬細胞(=F4/80+CD206+)。此結果亦與在小鼠異種移植物模型中觀察到之腫瘤生長抑制一致。 The above data also demonstrate that CBP501, when combined with CDDP or CDDP plus anti-PD1 antibody, can statistically significantly reduce M2 macrophages (=F4/80+CD206+) that support tumor growth and immune escape. This result is also consistent with the tumor growth inhibition observed in the mouse xenograft model.

<110> 日商坎巴斯有限公司(CanBas Co.,Ltd.) <110> CanBas Co.,Ltd.

<120> 用於癌症治療之肽及擬肽與T細胞活化劑及/或查核點抑制劑之組合 <120> Combinations of peptides and peptidomimetics with T cell activators and/or checkpoint inhibitors for cancer therapy

<130> C86869/200859 <130> C86869/200859

<140> 105134194 <140> 105134194

<141> 2016-10-21 <141> 2016-10-21

<150> US 62/245,899 <150> US 62/245,899

<151> 2015-10-23 <151> 2015-10-23

<150> US 62/345,416 <150> US 62/345,416

<151> 2016-06-03 <151> 2016-06-03

<160> 139 <160> 139

<170> PatentIn version 3.5 <170> PatentIn version 3.5

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<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

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<222> (11)..(12) <222> (11)..(12)

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<213> 人工序列 <213> Artificial sequence

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<223> 合成肽 <223> Synthetic peptides

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<222> (4)..(4) <222> (4)..(4)

<223> d-或l-Trp <223> d- or l-Trp

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<222> (5)..(5) <222> (5)..(5)

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<222> (6)..(6) <222> (6)..(6)

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<222> (9)..(9) <222> (9)..(9)

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<213> 人工序列 <213> Artificial sequence

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<223> 合成肽 <223> Synthetic peptides

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<222> (5)..(5) <222> (5)..(5)

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<222> (11)..(12) <222> (11)..(12)

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<213> 人工序列 <213> Artificial sequence

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<221> MOD_RES <221>MOD_RES

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<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<223> d-或l-Arg <223> d- or l-Arg

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<222> (8)..(8) <222> (8)..(8)

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<221> MOD_RES <221>MOD_RES

<222> (9)..(9) <222> (9)..(9)

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<221> MOD_RES <221>MOD_RES

<222> (10)..(12) <222> (10)..(12)

<223> d-或l-Arg <223> d- or l-Arg

<400> 46

<400> 46

<210> 47 <210> 47

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(3) <222> (1)..(3)

<223> d-或l-Arg <223> d- or l-Arg

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<221> MOD_RES <221>MOD_RES

<222> (4)..(4) <222> (4)..(4)

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<221> MOD_RES <221>MOD_RES

<222> (5)..(6) <222> (5)..(6)

<223> d-或l-Arg <223> d- or l-Arg

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<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

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<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

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<221> MOD_RES <221>MOD_RES

<222> (9)..(9) <222> (9)..(9)

<223> Xaa係任一胺基酸 <223> Any amino acid of Xaa series

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (10)..(10) <222> (10)..(10)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係任一胺基酸 <223> Any amino acid of Xaa series

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (12)..(12) <222> (12)..(12)

<400> 47

<400> 47

<210> 48 <210> 48

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(3) <222> (1)..(3)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(4) <222> (4)..(4)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (5)..(6) <222> (5)..(6)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

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<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係任一胺基酸 <223> Any amino acid of Xaa series

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (9)..(9) <222> (9)..(9)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (10)..(10) <222> (10)..(10)

<223> Xaa係任一胺基酸 <223> Any amino acid of Xaa series

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (11)..(11) <222> (11)..(11)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (12)..(12) <222> (12)..(12)

<400> 48

<400> 48

<210> 49 <210> 49

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(2) <222> (1)..(2)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(6) <222> (4)..(6)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (9)..(9) <222> (9)..(9)

<223> Xaa係任一胺基酸 <223> Any amino acid of Xaa series

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (10)..(10) <222> (10)..(10)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係任一胺基酸 <223> Any amino acid of Xaa series

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (12)..(12) <222> (12)..(12)

<400> 49

<400> 49

<210> 50 <210> 50

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(2) <222> (1)..(2)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(6) <222> (4)..(6)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係任一胺基酸 <223> Any amino acid of Xaa series

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (9)..(9) <222> (9)..(9)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (10)..(10) <222> (10)..(10)

<223> Xaa係任一胺基酸 <223> Any amino acid of Xaa series

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係(Phe-2,3,4,5,6-F)、(Phe-3,4,5F)或(Phe-4CF3) <223> Xaa line (Phe-2,3,4,5,6-F), (Phe-3,4,5F) or (Phe-4CF3)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (12)..(12) <222> (12)..(12)

<400> 50

<400> 50

<210> 51 <210> 51

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(2) <222> (1)..(2)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(6) <222> (4)..(6)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

<400> 51

<400> 51

<210> 52 <210> 52

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(2) <222> (1)..(2)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<223> (d-或l-Gln)或(d-或l-Arg) <223> (d-or l-Gln) or (d-or l-Arg)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(4) <222> (4)..(4)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (5)..(5) <222> (5)..(5)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (6)..(6) <222> (6)..(6)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係d-或l-Arg <223> Xaa series d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (9)..(9) <222> (9)..(9)

<400> 52

<400> 52

<210> 53 <210> 53

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(1) <222> (1)..(1)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (2)..(2) <222> (2)..(2)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(5) <222> (3)..(5)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (6)..(6) <222> (6)..(6)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

<223> d-或l-Arg <223> d- or l-Arg

<400> 53

<400> 53

<210> 54 <210> 54

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(1) <222> (1)..(1)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (2)..(2) <222> (2)..(2)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(4) <222> (4)..(4)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (5)..(5) <222> (5)..(5)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (6)..(6) <222> (6)..(6)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(9) <222> (8)..(9)

<223> d-或l-Arg <223> d- or l-Arg

<400> 54

<400> 54

<210> 55 <210> 55

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(1) <222> (1)..(1)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係(d-或l-Phe-2,3,4,5,6-F) <223> Xaa series (d- or l-Phe-2,3,4,5,6-F)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<223> d-或l-Ser <223> d- or l-Ser

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(4) <222> (4)..(4)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (5)..(5) <222> (5)..(5)

<223> d-或l-Ser <223> d- or l-Ser

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (6)..(6) <222> (6)..(6)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(9) <222> (7)..(9)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (10)..(10) <222> (10)..(10)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (11)..(12) <222> (11)..(12)

<223> d-或l-Arg <223> d- or l-Arg

<400> 55

<400> 55

<210> 56 <210> 56

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(2) <222> (1)..(2)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(6) <222> (4)..(6)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

<223> d-或l-Ser <223> d- or l-Ser

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (9)..(9) <222> (9)..(9)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (10)..(10) <222> (10)..(10)

<223> d-或l-Ser <223> d- or l-Ser

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係(d-或l-Phe-2,3,4,5,6-F) <223> Xaa series (d- or l-Phe-2,3,4,5,6-F)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (12)..(12) <222> (12)..(12)

<400> 56

<400> 56

<210> 57 <210> 57

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(1) <222> (1)..(1)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(4) <222> (4)..(4)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (5)..(5) <222> (5)..(5)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (6)..(6) <222> (6)..(6)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係(d-或l-Phe-2,3,4,5,6-F) <223> Xaa series (d- or l-Phe-2,3,4,5,6-F)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (9)..(9) <222> (9)..(9)

<400> 57

<400> 57

<210> 58 <210> 58

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (1)..(1) <222> (1)..(1)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係(d-或l-Phe-2,3,4,5,6-F) <223> Xaa series (d- or l-Phe-2,3,4,5,6-F)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (3)..(3) <222> (3)..(3)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (4)..(4) <222> (4)..(4)

<223> d-或l-Trp <223> d- or l-Trp

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (5)..(5) <222> (5)..(5)

<223> d-或l-Arg <223> d- or l-Arg

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (6)..(6) <222> (6)..(6)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (7)..(7) <222> (7)..(7)

<220> <220>

<221> MOD_RES <221>MOD_RES

<222> (8)..(9) <222> (8)..(9)

<223> d-或l-Arg <223> d- or l-Arg

<400> 58

<400> 58

<210> 59 <210> 59

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 59

<400> 59

<210> 60 <210> 60

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 60

<400> 60

<210> 61 <210> 61

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 61

<400> 61

<210> 62 <210> 62

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 62

<400> 62

<210> 63 <210> 63

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 63

<400> 63

<210> 64 <210> 64

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 64

<400> 64

<210> 65 <210> 65

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 65

<400> 65

<210> 66 <210> 66

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 66

<400> 66

<210> 67 <210> 67

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 67

<400> 67

<210> 68 <210> 68

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 68

<400> 68

<210> 69 <210> 69

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 69

<400> 69

<210> 70 <210> 70

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 70

<400> 70

<210> 71 <210> 71

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (9)..(9) <222> (9)..(9)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 71

<400> 71

<210> 72 <210> 72

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 72

<400> 72

<210> 73 <210> 73

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (9)..(9) <222> (9)..(9)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (10)..(10) <222> (10)..(10)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 73

<400> 73

<210> 74 <210> 74

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 74

<400> 74

<210> 75 <210> 75

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (9)..(9) <222> (9)..(9)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 75

<400> 75

<210> 76 <210> 76

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 76

<400> 76

<210> 77 <210> 77

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 77

<400> 77

<210> 78 <210> 78

<211> 17 <211> 17

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (10)..(10) <222> (10)..(10)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 78

<400> 78

<210> 79 <210> 79

<211> 15 <211> 15

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 79

<400> 79

<210> 80 <210> 80

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 80

<400> 80

<210> 81 <210> 81

<211> 6 <211> 6

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 81

<400> 81

<210> 82 <210> 82

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 82

<400> 82

<210> 83 <210> 83

<211> 11 <211> 11

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 83

<400> 83

<210> 84 <210> 84

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 84

<400> 84

<210> 85 <210> 85

<211> 11 <211> 11

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 85

<400> 85

<210> 86 <210> 86

<211> 6 <211> 6

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<400> 86

<400> 86

<210> 87 <210> 87

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 87

<400> 87

<210> 88 <210> 88

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 88

<400> 88

<210> 89 <210> 89

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe4NO2 <223> Xaa series d-Phe4NO2

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 89

<400> 89

<210> 90 <210> 90

<211> 11 <211> 11

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 90

<400> 90

<210> 91 <210> 91

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 91

<400> 91

<210> 92 <210> 92

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 92

<400> 92

<210> 93 <210> 93

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(2) <222> (1)..(2)

<223> d-Arg <223> d-Arg

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> d-Gln <223> d-Gln

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(6) <222> (4)..(6)

<223> d-Arg <223> d-Arg

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (9)..(9) <222> (9)..(9)

<223> d-Ser <223> d-Ser

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (10)..(10) <222> (10)..(10)

<223> d-Trp <223> d-Trp

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (11)..(11) <222> (11)..(11)

<223> d-Ser <223> d-Ser

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 93

<400> 93

<210> 94 <210> 94

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(2) <222> (1)..(2)

<223> d-Arg <223> d-Arg

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> d-Gln <223> d-Gln

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(6) <222> (4)..(6)

<223> d-Arg <223> d-Arg

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> d-Ser <223> d-Ser

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (9)..(9) <222> (9)..(9)

<223> d-Trp <223> d-Trp

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (10)..(10) <222> (10)..(10)

<223> d-Ser <223> d-Ser

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 94

<400> 94

<210> 95 <210> 95

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<400> 95

<400> 95

<210> 96 <210> 96

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 96

<400> 96

<210> 97 <210> 97

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (9)..(9) <222> (9)..(9)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 97

<400> 97

<210> 98 <210> 98

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (9)..(9) <222> (9)..(9)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (10)..(10) <222> (10)..(10)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 98

<400> 98

<210> 99 <210> 99

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (9)..(9) <222> (9)..(9)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 99

<400> 99

<210> 100 <210> 100

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (11)..(11) <222> (11)..(11)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 100

<400> 100

<210> 101 <210> 101

<211> 6 <211> 6

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 101

<400> 101

<210> 102 <210> 102

<211> 7 <211> 7

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 102

<400> 102

<210> 103 <210> 103

<211> 7 <211> 7

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 103

<400> 103

<210> 104 <210> 104

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 104

<400> 104

<210> 105 <210> 105

<211> 19 <211> 19

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (13)..(13) <222> (13)..(13)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 105

<400> 105

<210> 106 <210> 106

<211> 18 <211> 18

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (13)..(13) <222> (13)..(13)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 106

<400> 106

<210> 107 <210> 107

<211> 11 <211> 11

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 107

<400> 107

<210> 108 <210> 108

<211> 14 <211> 14

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 108

<400> 108

<210> 109 <210> 109

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 109

<400> 109

<210> 110 <210> 110

<211> 16 <211> 16

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (12)..(12) <222> (12)..(12)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (13)..(13) <222> (13)..(13)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (14)..(14) <222> (14)..(14)

<223> Xaa係1-胺基十一酸 <223> Xaa series 1-aminoundecanoic acid

<400> 110

<400> 110

<210> 111 <210> 111

<211> 19 <211> 19

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (8)..(8) <222> (8)..(8)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 111

<400> 111

<210> 112 <210> 112

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 112

<400> 112

<210> 113 <210> 113

<211> 14 <211> 14

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 113

<400> 113

<210> 114 <210> 114

<211> 16 <211> 16

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係1-胺基十一酸 <223> Xaa series 1-aminoundecanoic acid

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 114

<400> 114

<210> 115 <210> 115

<211> 15 <211> 15

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係1-胺基十一酸 <223> Xaa series 1-aminoundecanoic acid

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 115

<400> 115

<210> 116 <210> 116

<400> 116 <400> 116

000 000

<210> 117 <210> 117

<211> 14 <211> 14

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係1-胺基十一酸 <223> Xaa series 1-aminoundecanoic acid

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 117

<400> 117

<210> 118 <210> 118

<400> 118 <400> 118

000 000

<210> 119 <210> 119

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係1-胺基十一酸 <223> Xaa series 1-aminoundecanoic acid

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 119

<400> 119

<210> 120 <210> 120

<211> 9 <211> 9

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係1-8-胺基辛酸 <223> Xaa series 1-8-aminooctanoic acid

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 120

<400> 120

<210> 121 <210> 121

<400> 121 <400> 121

000 000

<210> 122 <210> 122

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 122

<400> 122

<210> 123 <210> 123

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 123

<400> 123

<210> 124 <210> 124

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 124

<400> 124

<210> 125 <210> 125

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係1-8-胺基辛酸 <223> Xaa series 1-8-aminooctanoic acid

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<400> 125

<400> 125

<210> 126 <210> 126

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係1-8-胺基辛酸 <223> Xaa series 1-8-aminooctanoic acid

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 126

<400> 126

<210> 127 <210> 127

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 127

<400> 127

<210> 128 <210> 128

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 128

<400> 128

<210> 129 <210> 129

<211> 13 <211> 13

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 129

<400> 129

<210> 130 <210> 130

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 130

<400> 130

<210> 131 <210> 131

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (2)..(2) <222> (2)..(2)

<223> Xaa係1-8-胺基辛酸 <223> Xaa series 1-8-aminooctanoic acid

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (3)..(3) <222> (3)..(3)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (4)..(4) <222> (4)..(4)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 131

<400> 131

<210> 132 <210> 132

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe4NO2 <223> Xaa series d-Phe4NO2

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 132

<400> 132

<210> 133 <210> 133

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe4NO2 <223> Xaa series d-Phe4NO2

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係2-萘基-丙胺醯基 <223> Xaa series 2-naphthyl-acrylamide

<400> 133

<400> 133

<210> 134 <210> 134

<211> 12 <211> 12

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係d-Phe4NO2 <223> Xaa series d-Phe4NO2

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe4NO2 <223> Xaa series d-Phe4NO2

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 134

<400> 134

<210> 135 <210> 135

<211> 7 <211> 7

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 135

<400> 135

<210> 136 <210> 136

<211> 8 <211> 8

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 136

<400> 136

<210> 137 <210> 137

<211> 6 <211> 6

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<400> 137

<400> 137

<210> 138 <210> 138

<211> 7 <211> 7

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (7)..(7) <222> (7)..(7)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 138

<400> 138

<210> 139 <210> 139

<211> 6 <211> 6

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 合成肽 <223> Synthetic peptides

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (1)..(1) <222> (1)..(1)

<223> Xaa係苯甲醯基-苯丙胺酸 <223> Xaa series benzoyl-phenylalanine

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (5)..(5) <222> (5)..(5)

<223> Xaa係d-Phe-2,3,4,5,6-F <223> Xaa series d-Phe-2,3,4,5,6-F

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE

<222> (6)..(6) <222> (6)..(6)

<223> Xaa係環己基-丙胺酸 <223> Xaa series cyclohexyl-alanine

<400> 139

<400> 139

Claims

A use of a peptide compound or a pharmaceutically acceptable salt thereof in combination with a platinum-containing drug and an immune checkpoint inhibitor for the preparation of a medicine for preventing or treating cell proliferation disorders in mammals, wherein the immune checkpoint inhibitor The agent comprises an anti-CTLA-4, anti-PD1, anti-PD-L1, anti-PD-L2, anti-VISTA, anti-TIM3, anti-LAG-3 or anti-BTLA antibody; wherein the peptide compound is selected from the group consisting of: (d- Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)( d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO: 80); (d-Arg)(d-Arg)(d-Arg)(d-Gln)(d- Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)( SEQ ID NO:94); and (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 1 ), which is attached or coupled to a cell penetrating molecule; and wherein the mammal has a white blood cell count of less than 10,000 white blood cells per microliter of blood (wbc/μl).

The use according to claim 1, wherein the medicine contains a T cell activator or is used in combination with a T cell activator.

The use according to claim 2, wherein the T cell activator targets CD28, OX40, GITR, CD137, CD27 or HVEM.

The use according to claim 2, wherein the T cell activator comprises anti-CD28, anti-OX40, anti-GITR, anti-CD137, anti-CD27 or anti-HVEM antibodies.

The use according to claim 4, wherein the antibody comprises subsequences binding to CD28, OX40, GITR, CD137, CD27 or HVEM.

The use according to claim 1, wherein the antibody comprises a subsequence that binds to CTLA-4, PD1, PD-L1, PDL2, VISTA, TIM3, LAG-3 or BTLA.

The use according to claim 4, wherein the antibody comprises a bispecific antibody binding to one or more of the following: CD28, OX40, GITR, CD137, CD27 and HVEM.

The use according to claim 1, wherein the antibody comprises a bispecific antibody that binds to one or more of the following: CTLA-4, PD1, PD-L1, PDL2, VISTA, TIM3, LAG-3 and BTLA.

The use according to claim 1 or 4, wherein the antibody comprises a mammalian antibody.

The use according to claim 4, wherein the antibody comprises a human, humanized, primatized or chimeric antibody that binds to: CD28, OX40, GITR, CD137, CD27 or HVEM.

The use of claim 1, wherein the antibody comprises a human, humanized, primatized or chimeric antibody that binds to: CTLA-4, PD1, PD-L1, PDL2, VISTA, TIM3, LAG-3 Or BTLA.

The use according to claim 1 or 4, wherein the antibody or its subsequence comprises Fab, Fab', F(ab') ₂ , Fv, Fd, single-chain Fv (scFv), disulfide-bonded Fvs (sdFv), V _L , V _H , camelid Ig, V-NAR, VHH, trispecific (Fab ₃ ), bispecific (Fab ₂ ), diabody ((V _L -V _H ) ₂ or (V _H -V _L ) ₂ ), trivalent antibody (trivalent), tetravalent antibody (tetravalent), minibody ((scF _V _-CH 3 ) ₂ ), bispecific single chain Fv (bi-scFv), IgGdeltaCH2, scFv- Fc, (scFv) ₂ -Fc, affibody, aptamer, avimer or nanobody.

The use according to claim 1, wherein the mammal has a white blood cell count between 4,000 and 10,000 white blood cells per microliter of blood (wbc/μl).

The use of claim 1, wherein the mammal has a white blood cell count of less than 9,000 white blood cells per microliter of blood (wbc/μl).

The use according to claim 1, wherein the mammal has a white blood cell count between 4,000 and 9,000 white blood cells per microliter of blood (wbc/μl).

The use of claim 1, wherein the mammal has a white blood cell count of less than 8,000 white blood cells per microliter of blood (wbc/μl).

The use according to claim 1, wherein the mammal has less than 7,000 white blood cells per microliter of blood White blood cell count (wbc/μl).

The use according to claim 1, wherein the mammal has a white blood cell count which is less than the upper limit of normal value of white blood cells per microliter of blood (wbc/μl) per clinical laboratory.

The use according to claim 1, wherein the peptide compound or its pharmaceutically acceptable salt and/or the immune checkpoint inhibitor constitute a pharmaceutical formulation.

As the use of claim item 1, wherein the pharmaceutically acceptable salt is selected from acetate, sulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, hydrogen phosphate Salt, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, propionate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate , heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4- Di-acid salt, hexyne-1,6-dioic acid salt, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate salt, phthalate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate , tartrate, methane-sulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate.

The use according to claim 1, wherein the cell penetrating molecule is joined to the peptide compound by a covalent bond or a peptide or non-peptide linker.

The use according to claim 21, wherein the cell penetrating molecule comprises polar/charged amino acids and non- Alternative to polar, hydrophobic amino acids.

The use according to claim 21, wherein the cell penetrating molecule comprises a polycation or an amphipathic α-helical structure.

The use according to claim 21, wherein the peptide compound and/or the cell penetrating molecule comprises an L-isomer or a D-isomer amino acid or a combination of an L-isomer and a D-isomer amino acid mixture.

The use according to claim 21, wherein the cell penetrating molecule comprises a poly-arginine (Arg) sequence.

The use according to claim 1, wherein the peptide compound or a pharmaceutically acceptable salt thereof is administered before, together with or after the administration of the immune checkpoint inhibitor.

The use according to claim 2, wherein the peptide compound or a pharmaceutically acceptable salt thereof is administered before, together with or after the administration of the T cell activator.

The use according to any one of claims 1 to 4 and 13 to 27, wherein the cell proliferation disorder comprises tumor or cancer.

The use according to claim 28, wherein the cell proliferation disorder comprises metastatic tumor or cancer.

The use according to claim 28, wherein the tumor or cancer comprises lung tumor or lung cancer.

The use according to claim 28, wherein the tumor or cancer comprises epithelial carcinoma, sarcoma, lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma, glioblastoma, meningioma, neuroblastoma tumor, retinoblastoma, astrocytoma, oligodendroglioma, mesothelioma or reticuloendothelioma, lymphoid or hematopoietic neoplasia, neoplasm, cancer or malignancy.